Surface active agent compositions and methods for enhancing oxygenation, reducing bacteria and improving wound healing at a site of treatment

ABSTRACT

This present invention relates generally to the use of novel formulations comprising a surface active polymer to enhance oxygenation in skin and other soft tissue. The present invention also discloses formulations that can be used to improve clinical outlook and reducing bacteria. Methods of making and using the same are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/321,106, entitled “Surface Active AgentCompositions and Methods for Enhancing Oxygenation, Reducing Bacteriaand Improving Wound Healing at a Site of Treatment” filed on May 2,2012, which is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/US2010/035440 entitled “Surface ActiveAgent Compositions and Methods for Enhancing Oxygenation, ReducingBacteria and Improving Wound Healing at a Site of Treatment”, filed onMay 19, 2010, which claims priority to and benefit of U.S. ProvisionalApplication No. 61/179,577, entitled “Poloxamer Formulations and MethodsFor Enhancing Oxygenation, Reducing Bacteria and Improving Wound Healingat a Site of Treatment” filed on May 19, 2009, each of which is herebyincorporated by reference in its entirety.

GOVERNMENT INTERESTS

Not Applicable

PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable

BACKGROUND

Poloxamers are water-soluble triblock copolymers composed of hydrophilicpolyethylene oxide (PEO) and hydrophobic polypropylene oxide (PPO)blocks linked together. The amphiphilic nature of these block copolymerscan be varied by controlling the length of the PEO and/or PPO blockcomponents (Ahmed et al., 2001). Several members of this poloxamerfamily of chemicals (such as poloxamer 188 and 407) are known to bebiocompatible and non-toxic to mammalian cells and tissues, making themuseful for biomedical applications. These compounds are surface acting(or “surface active”) agents (i.e. “surfactants”) and known toincorporate into or onto mammalian cell membranes, and thereby reduceprotein adsorption and cell adhesion.

The skin serves as a protective barrier against the environment. Theskin serves as a barrier to infection and prevents the loss of water andelectrolytes from the body. Thus, the loss of the integrity of largeportions of the skin as a result of illness or injury can lead to majordisability or even death.

Every year in the United States there are 1.1 million burn patients whorequire medical attention and 6.5 million patients are reported to havechronic skin ulcers caused by pressure, venous stasis, or diabetesmellitus. Thus, acceleration of skin wound healing has been an activearea of medical research and improved designs of skin repair materialshave been sought for decades.

There is a long felt need in the art for compositions and methods usefulfor treating injuries and wounds topically. The present inventionaddresses the need by providing novel formulations and methods oftreating skin and other soft tissues that enhance oxygenation, reducebacteria at the treated site, improve healing, and any combinationthereof.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides methods ofincreasing oxygenation at a site on a subject comprising contacting asite with a first composition comprising at least one surface activeagent and optionally at least one additional therapeutic agent, whereinupon contacting the site with the composition the site has increasedoxygenation. In some embodiments the site has been identified as a siteneeding increased oxygenation. In some embodiments, the subject is asubject in need of increased oxygenation at said site. In someembodiments, the at least one surface active agent is a surface activecopolymer. In some embodiments, the composition comprises about 0-10%w/w of the surface active agent. In some embodiments, the compositioncomprises about 5% w/w of said surface active agent.

In some embodiments, the present invention provides compositions fortreating a site to increase oxygenation, reduce bacterial count, and/orincrease healing at the treated site on a subject comprising at leastone surface active agent and optionally at least one additionaltherapeutic agent. In some embodiments, the compositions are liquids orgels.

In some embodiments, the present invention provides fabric materialscomprising a composition comprising at least one surface active agentand optionally at least one additional therapeutic agent. In someembodiments, the at least one surface active agent is an surface activecopolymer. In some embodiments, the fabric materials are used toincrease oxygenations, reduce bacterial count, or improve wound healingat a site on a subject.

DESCRIPTION OF DRAWINGS

None

DETAILED DESCRIPTION

This invention relates generally to the use of novel formulationscomprising a surface active polymer to enhance oxygenation in skin andother soft tissue. The formulations can also be used to improve clinicaloutlook, including to improve healing and reduce bacteria.

As used herein, the term “composition” can also be referred to as aformulation. The composition can consist of one ingredient or comprisemore than one ingredients. The number of components or ingredients doesnot differentiate between a composition or a formulation. In someembodiments, the composition is a pharmaceutical composition. In someembodiments, the composition is a therapeutic composition.

The present invention is based on the discovery described herein thatthe administration (e.g. topical) of a composition comprising a surfaceactive copolymer such as a poloxamer can be used, in some embodiments,to increase oxygenation, reducing bacteria, and/or improving healing ata tissue site, and any combination thereof. In some embodiments, thepresent invention provides compositions and methods for increasingoxygenation, reducing bacteria, and/or improving healing at a treatedsite. In some embodiments, the compositions can be used to increaseoxygenation, reduce bacteria, and/or improve healing at the site ofspecific injuries or at sites associated with certain diseases andconditions. The compositions and methods can also benefit healthy skinand other soft tissue. Without wishing to be bound by theory, it ishypothesized that the surfactant component acts as an oxygen reservoirand/or delivery vehicle to increase oxygenation, reduce bacteria, andimprove healing at a treated site.

In some embodiments, the present invention provides methods of treatinga tissue site. In some embodiments, the method comprises applying (e.g.topically) to the tissue site a pharmaceutical composition comprising asurfactant and optionally at least one additional therapeutic agent. Insome embodiments, the amount of the composition that is applied is anamount effective to increase oxygenation, reduce bacteria and/or improvehealing at the tissue site. In some embodiments, the compositioncomprises at least one surface active copolymer and optionally at leastone additional therapeutic agent to increase oxygenation, reducebacteria and/or improve healing at the tissue site.

In some embodiments, the at least one surface active copolymer is apoloxamer, a meroxapol, or a poloxamine. In some embodiments, thepharmaceutical composition comprises poloxamer 188.

Disclosed herein are formulations of surfactants, for example,poloxamers or other surface active agents for delivery (e.g. topical) tohealthy, injured and/or diseased tissues. The formulations can be usedto enhance oxygenation, reduce bacteria and/or improve healing. In someembodiments, wounds (e.g. chronic or acute) treated with theformulations disclosed herein demonstrate increased oxygenation, orimproved clinical outlook, which can include improved healing and/orreduction in bacteria at the site of the wound.

In some embodiments, the present invention provides compositionscomprising at least one surface active copolymer and methods of treatinga site of injury (e.g., burn injury, chronic wounds, skin grafts orother injury to skin or soft tissue) or exposed soft tissue using thecompositions or formulations disclosed herein. In some embodiments, thesurface active copolymers include, but are not limited to, poloxamers,meroxapols, and poloxamines.

In some embodiments, a composition or formulation disclosed hereincomprises at least one surface active copolymer or surface active agentat about 0.01%-85% w/w. In some embodiments, a composition orformulation disclosed herein comprises at least one surface activecopolymer or surface active agent at about 0.01%-75% w/w. In someembodiments, a composition or formulation disclosed herein comprises atleast one surface active copolymer or surface active agent at about0.01%-0.1%, about 0.01%-0.5%, or about 0.01%-1.0% w/w. In someembodiments, a composition or formulation disclosed herein comprises atleast one surface active copolymer or surface active agent at about0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%,about 0.07%, about 0.08%, or about 0.09% w/w. In some embodiments, acomposition or formulation disclosed herein comprises at least onesurface active copolymer or surface active agent at about 0.01%-60% w/w,about 0.01%-50% w/w, about 0.01%-50% w/w, about 0.01%-40% w/w, about0.01%-30% w/w, about 0.01%-20% w/w, about 0.01%-10% w/w, about 0.01%-5%w/w, about 0.01%-3%, 0.5%-60% w/w, about 0.5%-50% w/w, about 0.5%-50%w/w, about 0.5%-40% w/w, about 0.5%-30% w/w, about 0.5%-20% w/w, about0.5%-10% w/w, about 0.5%-5% w/w, about 0.5%-3% w/w, about 5%-10% w/w,about 4%-6% w/w, about 4%-5% w/w, about 5%-15% w/w, about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about0.8%, about 0.9%, about 1% w/w, about 2% w/w, about 3% w/w, about 4%w/w, about 5% w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9%w/w, or about 10% w/w.

Additional components optionally may be present in the compositions orformulations in the ranges shown in the table below, with the balancebeing supplied by water or another solvent. Appropriate ingredientsubstitutions may be made based on the type of ingredient, as would beunderstood by one of skill in the art.

Aloe Barbadensis Leaf Extract 0-30% Anti-inflammatory LinoleamidopropylPg-Dimonium 0-20% Surfactant & Chloride Phosphate moisturizerPolysorbate 20 0-10% Surfactant Sodium Coco-Pg Dimonium 0-30% Surfactant& Chloride Phosphate moisturizer Allantoin 0-20% Moisturizer DisodiumEDTA  0-5% Surfactant Sodium Benzoate  0-5% Preservative BenzalkoniumChloride  0-5% Preservative Fragrance  0-5% Fragrance IodopropynylButylcarbamate  0-5% Preservative Poloxamer 188 0.01-85%   Surfactant

In some embodiments, the composition comprises about 0-30%, about 0-20,about 0-15, about 0-10, about 0-5, about 5-10, about 15-20, about 10-15,about 1-3, about 3-5, about 5-7, about 7-9, about 9-11, about 1, about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, about 15, about 16, about17, about 18, about 19, about 20, about 21, about 22, about 23, about24, about 25, about 26, about 27, about 28, about 29, about 30% w/w Aloebarbadensis leaf extract.

In some embodiments, the composition comprises about 0-20, about 0-15,about 0-10, about 0-5, about 5-10, about 15-20, about 10-15, about 1-3,about 3-5, about 5-7, about 7-9, about 9-11, about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, or about 20% w/w linoleamidopropyl pg-dimonium chloridephosphate.

In some embodiments, the composition comprises about 0-10, about 0-5,about 0-4, about 0-3, about 0-2, about 0-1, less than 1, about 5-10,about 1-3, about 3-5, about 5-7, about 7-9, about 9-10, about 1, about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, orabout 10% w/w polysorbate 20.

In some embodiments, the composition comprises about 0-30%, about 0-20,about 0-15, about 0-10, about 0-5, about 0-4, about 0-3, about 0-2,about 0-1, less than 1, about 5-10, about 15-20, about 10-15, about 1-3,about 3-5, about 5-7, about 7-9, about 9-11, about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30% w/w sodium coco-pgdimonium chloride phosphate.

In some embodiments, the composition comprises about 0-20, about 0-15,about 0-10, about 0-5, about 0-4, about 0-3, about 0-2, about 0-1, about5-10, about 15-20, about 10-15, about 1-3, about 3-5, about 5-7, about7-9, about 9-11, about 1, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19, or about 20% w/wallantoin.

In some embodiments, the composition comprises about 0-5, about 0-4,about 0-3, about 0-2, about 0-1, less than 1, about 1-3, about 3-5,about 1, about 2, about 3, about 4, or about 5% w/w disodium EDTA.

In some embodiments, the composition comprises about 0-5, about 0-4,about 0-3, about 0-2, about 0-1, less than 1, about 1-3, about 3-5,about 1, about 2, about 3, about 4, or about 5% w/w sodium benzoate.

In some embodiments, the composition comprises about 0-5, about 0-4,about 0-3, about 0-2, about 0-1, less than 1, about 1-3, about 3-5,about 1, about 2, about 3, about 4, or about 5% w/w benzalkoniumchloride.

In some embodiments, the composition comprises about 0-5, about 0-4,about 0-3, about 0-2, about 0-1, less than 1, about 1-3, about 3-5,about 1, about 2, about 3, about 4, or about 5% w/w fragrance.

In some embodiments, the composition comprises about 0-5, about 0-4,about 0-3, about 0-2, about 0-1, less than 1, about 1-3, about 3-5,about 1, about 2, about 3, about 4, or about 5% w/w iodopropynylbutylcarbamate.

The therapeutic compositions of the invention may be formulated, forexample, as liquids or as stable gels.

In some embodiments, the compositions or formulations disclosed hereinare used to treat various injuries, diseases, and disorders which arecharacterized by reduced oxygen levels or that would benefit fromincreased oxygen levels. Examples of various injuries, diseases, anddisorders which are characterized by reduced oxygen levels or that wouldbenefit from increased oxygen levels include, but are not limited to,thermal injury, skin injury, soft tissue injury, non-healing skin wound,burns, acute wound, chronic wound, scrape, cut, incision, laceration,decubitis, pressure ulcer, chronic venous ulcer, venous stasis ulcer,diabetic ulcer, arterial ulcer, radiation ulcer, traumatic wound, opencomplicated non-healing wound, body piercing, bite wound, insect bite,insect sting, stab wound, gunshot wound, stretch injury, crush wound,compression wound, fracture, sprain, strain, stroke, infarction,aneurism, herniation, ischemia, fistula, dislocation, radiation,surgery, cell, tissue or organ grafting, and cancer.

In some embodiments, the present invention provides compositions andmethods for increasing oxygenation, reducing bacteria and/or improvinghealing of tissue a site treated by administering an effective amount ofa composition or formulation describes herein to a site (e.g., a site ofinjury). Examples of injuries, including burns include, but are notlimited to thermal burns, radiation burns, chemical burns, electricalburns, steam burns, and sunburn. In some embodiments, the burn is athermal injury. In some embodiments, the thermal injury is a cutaneousinjury or an injury of the mesentery of the intestine.

In some embodiments, the composition or formulation comprises at least1, at least 2, at least 3, at least 4, or at least 5 surface activecopolymers. In some embodiments, the composition or formulationcomprises 1, 2, 3, 4, or 5 surface active copolymers.

In some embodiments, the invention provides compositions or formulationsand methods for increasing oxygenation, reducing bacteria, and/orimproving healing in chronic wounds by administering an effective amountof the compositions or formulations disclosed herein to a site of achronic wound. Chronic wounds include, for example, but are not limitedto, venous stasis ulcers, diabetic wounds, arterial ulcers, and pressureulcers.

In some embodiments, the present invention provides compositions orformulations and methods for increasing oxygenation, reducing bacteria,and/or improving healing in skin or tissue following a surgicalprocedure, for example, skin grafts, microvascular surgery and tissueflaps, by administering an effective amount of the compositions orformulations disclosed herein to the site.

The route of administration or method of application can vary dependingon the formulation or composition being administered as well as on thesite of injury, disease, or disorder being treated. In some embodiments,any method of topical administration of the composition or formulationsdisclosed herein can be used to treat the injuries, diseases, anddisorders disclosed herein. In some embodiments, the compositions orformulations can be administered via routes, including, but not limitedto, direct, topical, cutaneous, mucosal, nasal, inhalation, oral, andophthalmic. Methods of applying or administration include, but are notlimited to contacting the site with a dressing material, extruder,aerosol, spray delivery, iontophoresis, a patch, a wipe, or atransdermal patch. The dressing material, extruder, aerosol, spraydelivery, iontophoresis, a patch, a wipe, or a transdermal patch cancomprise any composition or formulation described herein.

In some embodiments, the compositions, formulations, additionaltherapeutic agent, or another compound are administered as acontrolled-release formulation.

The dosage of the active compound(s) being administered will depend onthe condition being treated, the particular compound, and other clinicalfactors such as age, sex, weight, and health of the subject beingtreated, the route of administration of the compound(s), and the type ofcomposition being administered (gel, liquid, solution, suspension,aerosol, ointment, lotion, cream, paste, liniment, etc.). It is to beunderstood that the present invention has application for both human andveterinary use.

In some embodiments, the compositions or formulations described can beadministered at different times before and after an injury or surgicalprocedure, as well as varying the optional additional therapeutic agentsand the surface active copolymers.

In some embodiments, the composition or formulation described hereincomprises at least one poloxamer or other surface active copolymer agentat a concentration ranging from about 0.01% to about 85% w/w or at other% w/w as disclosed herein. Examples of poloxamers include, but are notlimited to, poloxamer-101, -105, -105 benzoate, -108, -122, -123, -124,-181, -182, -182 dibenzoate, -183, -184, -185, -188, -212, -215, -217,-231, -234, -235, -237, -238, -282, -284, -288, -331, -333, -334, -335,-338, -401, -402, -403, and -407. In some embodiments, the poloxamer ispoloxamer-188. In some embodiments, the poloxamer is poloxamer-407.

In some embodiments, the composition or formulation comprises poloxamer188 and benazlkonium chloride.

In some embodiments, the surface active copolymers is a meroxapol.Exemplary meroxapols include, but are not limited to, meroxapol 105,108, 171, 172, 174, 178, 251, 252, 254, 258, 311, 312, and 314.

In some embodiments, the surface active copolymer is a poloxamine.Exemplary poloxamines include, but are not limited to, poloxamine 304,504, 701, 702, 704, 707, 901, 904, 908, 1101, 1102, 1104, 1301, 1302,1304, 1307, 1501, 1502, 1504, and 1508.

In some embodiments, the composition (e.g. therapeutic orpharmaceutical) is formulated as a liquid or stable gel. The copolymersize may range, for example, from an M_(n) of about 600 to about 20,000.In some embodiments, the copolymer size may range, for example, from anM_(n) of about 1,000 to about 10,000.

In some embodiments, the present invention encompasses a compositioncomprising a surface active copolymer (e.g., a poloxamer, poloxamine, ormeroxapol) at about 0.01% to about 85% w/w, or about 1% to about 65%, orabout 1% to about 50%, or about 5% to about 40%, or about 10% to about40%. The surface active copolymer can also be used at other % w/w asdisclosed herein.

The surface active copolymers may be prepared at different temperaturesdepending on the type of formulation or composition being prepared, theroute of administration, the site of administration, etc. In someembodiments, the surface active copolymer is prepared at a temperatureranging from about 0° F. to about 70° F. In some embodiments, thesurface active copolymer is prepared at a temperature ranging from about5° F. to about 50° F. In some embodiments, the surface active copolymeris prepared at a temperature ranging from about 10° F. to about 40° F.In some embodiments, the surface active copolymer is prepared at atemperature ranging from about −10° F. to about 70° F., −10° F. to about60° F., −10° F. to about 50° F., −10° F. to about 40° F., −10° F. toabout 30° F., −10° F. to about 20° F., −10° F. to about 10° F., −10° F.to about 0° F. In some embodiments, the surface active copolymer isprepared at a temperature that is less than or equal to 0° F. In someembodiments, the surface active copolymer is prepared at a temperaturethat is less than or equal to −1° F. In some embodiments, the surfaceactive copolymer is prepared at a temperature that is less than or equalto −5° F.

The composition may further comprise an effective amount of at least oneadditional therapeutic agent. In some embodiments, the at least oneadditional therapeutic agent can be useful for the type of injury,disease, or disorder being treated. Additional therapeutic agentsinclude, but are not limited to, anesthetic, analgesic, antimicrobial,steroid, growth factor, cytokine, and anti-inflammatory agents.Anesthetic agents include, but are not limited to, benzocaine,lidocaine, bupivocaine, dibucaine, mepivocaine, etidocaine, tetracaine,butanilicaine, and trimecaine. In some embodiments, at least oneadditional therapeutic agent is at least one analgesic. In someembodiments, the at least one additional therapeutic is present in anamount that is less than about 1% by weight of the composition. In someembodiments, the at least one additional therapeutic is present in anamount that is less than about 0.95% by weight of the composition. Insome embodiments, the at least one additional therapeutic is present inan amount that is less than about 0.75% by weight of the composition.

In some embodiments, the additional therapeutic agent is anantimicrobial agent. In some embodiments, the antimicrobial agent is anantibacterial agent. In some embodiments, the antimicrobial agent is anantifungal agent. In some embodiments, the antimicrobial agent is anantiviral agent. Antimicrobial agents include, but are not limited to,silver sulfadiazine, Nystatin, Nystatin/triamcinolone, Bacitracin,nitrofurazone, nitrofurantoin, a polymyxin (e.g., Colistin, Surfactin,Polymyxin E, and Polymyxin B), doxycycline, antimicrobial peptides(e.g., natural and synthetic origin), Neosporin (i.e., Bacitracin,Polymyxin B, and Neomycin), Polysporin (i.e., Bacitracin and PolymyxinB). Additional antimicrobials include topical antimicrobials (i.e.,antiseptics), examples of which include silver salts, iodine,benzalkonium chloride, alcohol, hydrogen peroxide, and chlorhexidine. Insome embodiments, the antimicrobial is an antimicrobial other thanNystatin. In some embodiment, the additional therapeutic agent isaspirin, pentoxifylline, clopidogrel bisulfate, or other angiogenic, ora rheologic active agent. In some embodiments, the composition comprisesa “sub-lethal” amount of an antimicrobial.

As used herein the terms “sub-lethal dose” or “sub-lethal amount” refersto an amount of an antimicrobial agent or additional therapeutic that isless than the standard therapeutically effective amount. In the contextof the invention described herein, a sub-lethal amount of anantimicrobial agent may effectively eradicate or inhibit the growth of amicroorganisms or pathogens at the site being treated. For example, asub-lethal amount of an antimicrobial agent in some embodiments may befrom about 10% to greater than about 50% less than the standardtherapeutically effective amount or 10%, 15%, 20%, 25%, 30%, 35%, 40%,45% or 50% less than the standard therapeutically effective amountapproved by a regulatory agency. In some embodiments, a sub-lethalamount may be greater than 50% less than the standard therapeuticallyeffective amount.

For example, a standard therapeutically effective amount of a silversulfadiazine is about 1.0% by weight when used in an antimicrobial creamor gel. However, when silver sulfadiazine is administered in combinationwith a surface active agent, the amount of silver sulfadiazine in thecomposition may be decreased below the standard therapeuticallyeffective amount to, for example, less than 1.0% by weight. Therefore, asub-lethal amount of silver sulfadiazine in some embodiments, may beless than 0.95% by weight, less than 0.90% by weight, less than 0.85% byweight, less than 0.80% by weight, less than 0.75% by weight, less than0.70% by weight, less than 0.65% by weight, less than 0.60% by weight orless than 0.55% by weight of the composition. In some embodiments, theamount of silver sulfadiazine may be less than 0.5% by weight of thecomposition.

In some embodiments, the present invention provides methods of treatinga site of injury on a subject comprising administering a composition orformulation as described herein to the subject in an amount effective toincrease oxygen at the treated site. In some embodiments, the subject orthe site is identified as needing to have increased oxygen at thetreated site prior to the administration of a composition or formulationdescribed herein. In some embodiments, the composition or formulationcomprises a poloxamer.

Depending on such things as the type of formulation or composition beingprepared, the location to which it is to be applied, and the type ofinjury, disease, or disorder being treated, other agents can be added tothe formulation or composition. For example, other additives mayinclude, a moisturizer, a humectant, a demulcent, oil, water, anemulsifier, a thickener, a thinner, an additional surface active agent,a fragrance, a preservative, an antioxidant, a hydrotropic agent, achelating agent, a vitamin, a mineral, a permeation enhancer, a cosmeticadjuvant, a bleaching agent, a depigmentation agent, a foaming agent, aconditioner, a viscosifier, a buffering agent, and/or a sunscreen.

In some embodiments, the present invention provides administering cells,e.g., to a site of injury, disease, or disorder being treated. In someembodiments, the cells are part of the composition being administered.In some embodiments, the cells are applied separately (e.g. prior to orafter the composition has been administered). Examples of cells include,but are not limited to, stem cells, pluripotent stem cells, committedstem cells, embryonic stem cells, adult stem cells, bone marrow stemcells, adipose stem cells, umbilical cord stem cells, dura mater stemcells, precursor cells, differentiated cells, osteoblasts, myoblasts,neuroblasts, fibroblasts, glioblasts, germ cells, hepatocytes,chondrocytes, keratinocytes, melanocytes, smooth muscle cells, cardiacmuscle cells, connective tissue cells, glial cells, epithelial cells,endothelial cells, hormone-secreting cells, cells of the immune system,normal cells, Schwann cells, and neurons. In some embodiments, it isunnecessary to pre-select the type of stem cell that is to be used,because, in part, many types of stem cells can be induced todifferentiate in an organ specific pattern once delivered to a givenorgan or tissue. In some embodiments, the type of stem cell ispre-selected. In some embodiments, at least two different cells areused.

In some embodiments, the present invention provides methods foridentifying compounds which are useful for treating decreased oxygenlevels associated with an injury, disease, or condition. In someembodiments, the present invention provides methods for identifyingagents that can enhance the usage of the formulations and compositionsdescribed herein. For example, one can contact a site with a compositionor formulation described herein to increase oxygen levels at the site. Atest agent can be contacted at the site and the level of oxygenation isdetermined. If the level of oxygenation is increased then the test agentis said to enhance the formulation or composition's ability to increaseoxygenation. The same method can be used to determine agents thatinhibit the ability to increase oxygenation. The same method can bemodified to test for agents that can enhance or inhibit bacterial growthor colonization at the site. In contrast to the method for enhancingoxygenation, the method can be used to determine bacteria at the site.Methods of determining bacterial growth or oxygenation are well known toone of skill in the art and any method can be used. Reduction inbacterial can be determined by determining the number of bacteria thatare present at the site before and after contact with a composition. Forexample, the number of bacteria can be determined by obtaining a samplefrom the site and culturing the site to determine the number ofbacteria. The number of bacteria can be determined by, for example,colony formation assays, optical density, and the like. A reduction innumber indicates that the composition has reduced the bacteria count atthe site. In some embodiments, the bacterial count is reduced by atleast 5, 10, 15, 20, 25, 30, 40, 45, or 50%. In some embodiments, thebacterial count is reduced by about 5 to about 25%.

As used herein “improved healing” can refer to increased healing or thehealing rate that would occur without the presence of a compositiondisclosed herein. Healing can be measured, for example, by viewing thewound and determining the rate of wound area reduction. The improvedhealing can also be determined by determining the amount of wound areareduction. Wound area reduction can be determined using any system ormethod including, but not limited to, the Visitrak™ system (Smith &Nephew), a wound measurement device. In some embodiments, the wound areais reduced by at least 5, 10, 15, 20, 30, 40, or 50%.

The present invention further provides kits for administeringpharmaceutical compositions of the invention to subjects in needthereof. The methods described herein can be also be used for treatingsubjects in need of treatment of the various disorders, conditions, anddiseases.

Abbreviations and Acronyms

ASC—adipose tissue-derived stem cell

ECM—extracellular matrix

ES—embryonic stem cell

FACS—fluorescent activated cell sorting

FBS—fetal bovine serum

FGF—fibroblast growth factor

gf—growth factor

HSC—hematopoietic stem cell

IL-1β—interleukin-1 beta

PDGF—platelet-derived growth factor

PLA—processed lipoaspirate cells

SCGF-β—stem cell growth factor-β

SFM—serum-free medium

TNFα—tumor necrosis factor alpha

VEGF—Vascular endothelial growth factor

The following terminology will be used in accordance with thedefinitions set forth below. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Additionally any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of themethods, compositions, or formulations described herein.

Specific values listed below for radicals, substituents, and ranges arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

As used herein, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%. In some embodiments, the term “about”means plus or minus 20% of the numerical value of the number with whichit is being used. Therefore, about 50% can mean in the range of 45%-55%.Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbersand fractions thereof are presumed to be modified by the term “about.”One of skill in the art would also understand that where the presentspecification refers to a value with the term about, this is alsodiscloses the specific value (e.g. about 5 discloses 5).

The terms “additional therapeutically active compound” or “additionaltherapeutic agent,” as used herein, refers to the use or administrationof a compound for an additional therapeutic use for a particular injury,disease, or disorder being treated. Such a compound, for example, couldinclude one being used to treat an unrelated disease or disorder, or adisease or disorder which may not be responsive to the primary treatmentfor the injury, disease or disorder being treated. The additionalcompounds may also be used to treat symptoms associated with the injury,disease or disorder, including, but not limited to, pain andinflammation.

Adipose-derived stem cells (ASC) or “adipose-derived stromal cells”refer to cells that originate from adipose tissue. “Adipose” refers toany fat tissue. The adipose tissue may be brown or white adipose tissue,derived from subcutaneous, omental/visceral, mammary, gonadal, or otheradipose tissue site. In some embodiments, the adipose is subcutaneouswhite adipose tissue. Such cells may comprise a primary cell culture oran immortalized cell line. The adipose tissue may be from any organismhaving fat tissue. In some embodiments, the adipose tissue is mammalian.In some embodiments, the adipose tissue is human. A convenient source ofadipose tissue is from liposuction surgery, however, the source ofadipose tissue or the method of isolation of adipose tissue is notcritical to the invention.

As use herein, the terms “administration of” and or “administering”refers to the providing a compound, composition, or formulation to asubject. In some embodiments, the subject is in need of treatment. Insome embodiments, a prodrug of a compound is administered.

The term “adult” as used herein, is meant to refer to any non-embryonicor non-juvenile subject. For example the term “adult adipose tissue stemcell,” refers to an adipose stem cell, other than that obtained from anembryo or juvenile subject.

Cells or tissue are “affected” by an injury, disease or disorder if thecells or tissue have an altered phenotype relative to the same cells ortissue in a subject not afflicted with the injury, disease, condition,or disorder. As used herein, an “agonist” is a composition of matterthat, when administered to a mammal such as a human, enhances or extendsa biological activity of interest. Such effect may be direct orindirect.

A disease, condition, or disorder is “alleviated” if the severity of asymptom of the disease or disorder, the frequency with which such asymptom is experienced by a patient, or both, are reduced.

As used herein, “alleviating an injury, disease or disorder symptom,”means reducing the frequency or severity of the symptom.

As used herein, an “analog” of a chemical compound is a compound that,by way of example, resembles another in structure but is not necessarilyan isomer (e.g., 5-fluorouracil is an analog of thymine).

An “antagonist” is a composition of matter that when administered to amammal such as a human, inhibits or impedes a biological activityattributable to the level or presence of an endogenous compound in themammal. Such effect may be direct or indirect.

The term “antimicrobial agents” as used herein refers to anynaturally-occurring, synthetic, or semi-synthetic compound orcomposition or mixture thereof, which is safe for human or animal use aspracticed in the methods of this invention, and is effective in killingor substantially inhibiting the growth of microbes. “Antimicrobial” asused herein, includes antibacterial, antifungal, and antiviral agents.

“Antiviral agent,” as used herein means a composition of matter which,when delivered to a cell, is capable of preventing replication of avirus in the cell, preventing infection of the cell by a virus, orreversing a physiological effect of infection of the cell by a virus.Antiviral agents are well known and described in the literature. By wayof example, AZT (zidovudine, Retrovir® Glaxo Wellcome Inc., ResearchTriangle Park, NC) is an antiviral agent which is thought to preventreplication of HIV in human cells.

The term “biocompatible,” as used herein, refers to a material that doesnot elicit a substantial detrimental response in the host.

The term “biodegradable,” as used herein, means capable of beingbiologically decomposed. A biodegradable material differs from anon-biodegradable material in that a biodegradable material can bebiologically decomposed into units which may be either removed from thebiological system and/or chemically incorporated into the biologicalsystem.

The term “biological sample,” as used herein, refers to samples obtainedfrom a living organism, including skin, hair, tissue, blood, plasma,cells, sweat, and urine.

The term “bioresorbable,” as used herein, refers to the ability of amaterial to be resorbed in vivo. “Full” resorption means that nosignificant extracellular fragments remain. The resorption processinvolves elimination of the original implant materials through theaction of body fluids, enzymes, or cells. Resorbed calcium carbonatemay, for example, be redeposited as bone mineral, or by being otherwisere-utilized within the body, or excreted. “Strongly bioresorbable,” asthe term is used herein, means that at least 80% of the total mass ofmaterial implanted is resorbed within one year. As used herein “burn” or“burns” refer to any detectable injury to tissue caused by energyapplied to the tissue. The terms “burn” or “burns” further refer to anyburning, or charring of the tissue, including thermal burns caused bycontact with flames, hot liquids, hot surfaces, and other sources ofhigh heat as well as steam, chemical burns, radiation, and electricalburns. First degree burns show redness; second degree burns showvesication; third degree burns show necrosis through the entire skin.Burns of the first and second degree are partial-thickness burns, thoseof the third degree are full-thickness burns.

The term “clearance,” as used herein refers to the physiological processof removing a compound or molecule, such as by diffusion, exfoliation,removal via the bloodstream, and excretion in urine, or via sweat orother fluid.

A “compound,” as used herein, refers to any type of substance or agentthat is commonly considered a drug, or a candidate for use as a drug, aswell as combinations and mixtures of the above.

A “control” subject is a subject having the same characteristics as atest subject, such as a similar type of dependence, etc. The controlsubject may, for example, be examined at precisely or nearly the sametime the test subject is being treated or examined. The control subjectmay also, for example, be examined at a time distant from the time atwhich the test subject is examined, and the results of the examinationof the control subject may be recorded so that the recorded results maybe compared with results obtained by examination of a test subject.

A “test subject” is a subject being treated.

“Cytokine,” as used herein, refers to intercellular signaling molecules,the best known of which are involved in the regulation of mammaliansomatic cells. A number of families of cytokines, both growth promotingand growth inhibitory in their effects, have been characterizedincluding, for example, interleukins, interferons, and transforminggrowth factors. A number of other cytokines are known to those of skillin the art. The sources, characteristics, targets and effectoractivities of these cytokines have been described.

As used herein, a “derivative” of a compound refers to a chemicalcompound that may be produced from another compound of similar structurein one or more steps, as in replacement of H by an alkyl, acyl, or aminogroup.

The use of the word “detect” and its grammatical variants is meant torefer to measurement of the species without quantification, whereas useof the word “determine” or “measure” with their grammatical variants aremeant to refer to measurement of the species with quantification. Theterms “detect” and “identify” are used interchangeably herein.

As used herein, a “detectable marker” or a “reporter molecule” is anatom or a molecule that permits the specific detection of a compoundcomprising the marker in the presence of similar compounds without amarker. Detectable markers or reporter molecules include, but are notlimited to, radioactive isotopes, antigenic determinants, enzymes,nucleic acids available for hybridization, chromophores, fluorophores,chemiluminescent molecules, electrochemically detectable molecules, andmolecules that provide for altered fluorescence polarization or alteredlight scattering.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. As used herein, normalaging is included as a disease. A “disorder” in an animal is a state ofhealth in which the animal is able to maintain homeostasis, but in whichthe animal's state of health is less favorable than it would be in theabsence of the disorder. Left untreated, a disorder does not necessarilycause a further decrease in the animal's state of health.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect, such as alleviating symptoms of a disease ordisorder. In the context of administering compounds in the form of acombination, such as multiple compounds, the amount of each compound,when administered in combination with another compound(s), may bedifferent from when that compound is administered alone. Thus, aneffective amount of a combination of compounds refers collectively tothe combination as a whole, although the actual amounts of each compoundmay vary. The term “more effective” means that the selected effect isalleviated to a greater extent by one treatment relative to the secondtreatment to which it is being compared.

As used herein, a “functional” molecule is a molecule in a form in whichit exhibits a property or activity by which it is characterized. Afunctional enzyme, for example, is one that exhibits the characteristiccatalytic activity by which the enzyme is characterized.

“Graft” refers to any free (unattached) cell, tissue, or organ fortransplantation. “Allograft” refers to a transplanted cell, tissue, ororgan derived from a different animal of the same species.

“Xenograft” refers to a transplanted cell, tissue, or organ derived froman animal of a different species.

The term “growth factor” as used herein means a bioactive molecule thatpromotes the proliferation of a cell or tissue. Examples of growthfactors include, but are not limited to, transforming growthfactor-alpha (TGF-α), transforming growth factor-beta (TGF-β),platelet-derived growth factors including the AA, AB and BB isoforms(PDGF), fibroblast growth factors (FGF), including FGF acidic isoforms 1and 2, FGF basic form 2, and FGF 4, 8, 9 and 10, nerve growth factors(NGF) including NGF 2.5s, NGF 7.0s and beta NGF and neurotrophins, brainderived neurotrophic factor, cartilage derived factor, bone growthfactors (BGF), basic fibroblast growth factor, insulin-like growthfactor (IGF), vascular endothelial growth factor (VEGF), EG-VEGF,VEGF-related protein, Bv8, VEGF-E, granulocyte colony stimulating factor(G-CSF), insulin like growth factor (IGF) I and II, hepatocyte growthfactor, glial neurotrophic growth factor, stem cell factor (SCF),keratinocyte growth factor (KGF), skeletal growth factor, bone matrixderived growth factors, and bone derived growth factors and mixturesthereof. Some growth factors may also promote differentiation of a cellor tissue. TGF, for example, may promote growth and/or differentiationof a cell or tissue.

The term “increased oxygenation,” as used herein, refers to increasedoxygen levels measured at a site being treated according to the methodsdescribed herein compared with the oxygen levels at the same site priorto being treated according to the methods described herein, or ascompared with the oxygen levels of a comparable site left untreated.Oxygenation of skin or the surfaces of other tissues can be measuredusing a saturimetry device (two very thin electrodes that are positionedon the skin) or other probe or system that measures oxygen tension oroxygen levels in tissue. Unless otherwise specified the method ofdetermining oxygenation can be any method used to measure oxygenation.In some embodiments, the oxygenation is increased by at least 10%, 20%,30%, 40%, or 50%. In some embodiments, the oxygenation is increased byabout 5 to 20%, 10 to 20%, or 5 to 15%.

As used herein “injecting or applying” includes administration of acompound of the invention by any number of routes and means including,but not limited to, topical, oral, buccal, intravenous, intramuscular,intra arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal means.

As used herein, “injury” generally refers to damage, harm, or hurt;usually applied to damage inflicted on the body by an external force. Asused herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound of the invention in thekit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders in a subject. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains theidentified compound invention or be shipped together with a containerwhich contains the identified compound. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the instructional material and the compound be used cooperativelyby the recipient.

As used herein, a “ligand” is a compound that specifically binds to atarget compound or molecule. A ligand “specifically binds to” or “isspecifically reactive with” a compound when the ligand functions in abinding reaction which is determinative of the presence of the compoundin a sample of heterogeneous compounds.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

“Permeation enhancement” and “permeation enhancers” as used hereinrelate to the process and added materials which bring about an increasein the permeability of skin to a poorly skin permeatingpharmacologically active agent, i.e., so as to increase the rate atwhich the drug permeates through the skin and enters the bloodstream.“Permeation enhancer” is used interchangeably with “penetrationenhancer”.

The term “pharmaceutical composition” shall mean a compositioncomprising at least one active ingredient, whereby the composition isamenable to investigation for a specified, efficacious outcome in amammal (for example, without limitation, a human). Those of ordinaryskill in the art will understand and appreciate the techniquesappropriate for determining whether an active ingredient has a desiredefficacious outcome based upon the needs of the artisan. In someembodiments, a pharmaceutical composition comprises the compounds,compositions or formulations described herein.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate compound or derivativecan be combined and which, following the combination, can be used toadminister the appropriate compound to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The term “prevent,” as used herein, means to stop something fromhappening, or taking advance measures against something possible orprobable from happening. In the context of medicine, “prevention”generally refers to action taken to decrease the chance of getting adisease or condition.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or injury or exhibits only earlysigns of the disease or injury for the purpose of decreasing the risk ofdeveloping pathology associated with the disease or injury. As usedherein, the term “purified” and like terms relate to an enrichment of amolecule or compound relative to other components normally associatedwith the molecule or compound in a native environment. The term“purified” does not necessarily indicate that complete purity of theparticular molecule has been achieved during the process. A “highlypurified” compound as used herein refers to a compound that is greaterthan 90% pure.

The term “regulate” refers to either stimulating or inhibiting afunction or activity of interest.

A “receptor” is a compound or molecule that specifically binds to aligand.

A “sample,” as used herein, refers to a biological sample from asubject, including, but not limited to, normal tissue samples, diseasedtissue samples, biopsies, blood, saliva, feces, semen, tears, and urine.A sample can also be any other source of material obtained from asubject which contains cells, tissues, or fluid of interest.

The term “skin,” as used herein, refers to the commonly used definitionof skin, e.g., the epidermis and dermis, and the cells, glands, mucosa,and connective tissue which comprise the skin.

By the term “specifically binds,” as used herein, is meant a moleculewhich recognizes and binds a specific molecule, but does notsubstantially recognize or bind other molecules in a sample, or it meansbinding between two or more molecules as in part of a cellularregulatory process, where said molecules do not substantially recognizeor bind other molecules in a sample.

The term “standard,” as used herein, refers to something used forcomparison. For example, it can be a known standard agent or compoundwhich is administered and used for comparing results when administeringa test compound, or it can be a standard parameter or function which ismeasured to obtain a control value when measuring an effect of an agentor compound on a parameter or function. “Standard” can also refer to an“internal standard”, such as an agent or compound which is added atknown amounts to a sample and which is useful in determining such thingsas purification or recovery rates when a sample is processed orsubjected to purification or extraction procedures before a marker ofinterest is measured. Internal standards are often but are not limitedto, a purified marker of interest which has been labeled, such as with aradioactive isotope, allowing it to be distinguished from an endogenoussubstance in a sample.

A “subject” of diagnosis or treatment is a mammal, including a human.

As used herein, a “subject in need thereof” is a patient, animal,mammal, or human, who will benefit from the method of this invention. Insome embodiments, the subject in need thereof is identified as needingtreatment.

A “surface active agent” or “surfactant” is a substance that has theability to reduce the surface tension of materials and enablepenetration into and through materials.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.

The term “symptom,” as used herein, refers to any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by the patient and indicative of disease. In contrast, asign is objective evidence of disease. For example, a bloody nose is asign. It is evident to the patient, doctor, nurse and other observers.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered. In some embodiments, theamount of a compound, such as the additional therapeutic, isadministered in an amount that is less than the therapeuticallyeffective amount when compared to the amount that the compound isadministered as a single agent. In some embodiments, the amount is 50%of a therapeutically effective amount when administered as a singleagent.

The term “thermal injury” is used interchangeably with “thermal burn”herein.

“Tissue” means (1) a group of similar cells united to perform a specificfunction; (2) a part of an organism consisting of an aggregate of cellshaving a similar structure and function; or (3) a grouping of cells thatare similarly characterized by their structure and function, such asmuscle or nerve tissue.

The term “topical application,” as used herein, refers to administrationto a surface, such as the skin. This term is used interchangeably with“cutaneous application” in the case of skin. A “topical application” isa “direct application”.

By “transdermal” delivery is meant delivery by passage of a drug throughthe skin or mucosal tissue and into the bloodstream. Transdermal alsorefers to the skin as a portal for the administration of drugs orcompounds by topical application of the drug or compound thereto.“Transdermal” is used interchangeably with “percutaneous.”

As used herein, the term “treating” may include prophylaxis of thespecific injury, disease, disorder, or condition, or alleviation of thesymptoms associated with a specific injury, disease, disorder, orcondition and/or preventing or eliminating said symptoms. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease. “Treating” is used interchangeably with“treatment” herein. As used herein “wound” or “wounds” may refer to anydetectable break in the tissues of the body, such as injury to skin orto an injury or damage, or to a damaged site associated with a diseaseor disorder. Although the terms “wound” and “injury” are not alwaysdefined exactly the same way, the use of one term herein, such as“injury”, is not meant to exclude the meaning of the other term.

Chemical Definitions

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo.

The term “haloalkyl” as used herein refers to an alkyl radical bearingat least one halogen substituent, for example, chloromethyl, fluoroethylor trifluoromethyl and the like.

The term “C1-C_(n) alkyl” wherein n is an integer, as used herein,represents a branched or linear alkyl group having from one to thespecified number of carbon atoms. Typically, C1-C₆ alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like.

The term “C2-C_(n) alkenyl” wherein n is an integer, as used herein,represents an olefinically unsaturated branched or linear group havingfrom two to the specified number of carbon atoms and at least one doublebond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,and the like. The term “C2-C_(n) alkynyl” wherein n is an integer refersto an unsaturated branched or linear group having from two to thespecified number of carbon atoms and at least one triple bond. Examplesof such groups include, but are not limited to, 1-propynyl, 2-propynyl,1-butyryl, 2-butyryl, 1-pentynyl, and the like.

The term “C3-C_(n) cycloalkyl” wherein n=8, represents cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein the term “aryl” refers to an optionally substituted mono-or bicyclic carbocyclic ring system having one or two aromatic ringsincluding, but not limited to, phenyl, benzyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl, and the like. “Optionallysubstituted aryl” includes aryl compounds having from zero to foursubstituents, and “substituted aryl” includes aryl compounds having oneor more substituents. The term (C5-C8 alkyl)aryl refers to any arylgroup which is attached to the parent moiety via the alkyl group.

The term “bicyclic” represents either an unsaturated or saturated stable7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The term “heterocyclic group” refers to an optionally substituted mono-or bicyclic carbocyclic ring system containing from one to threeheteroatoms wherein the heteroatoms are selected from the groupconsisting of oxygen, sulfur, and nitrogen.

As used herein the term “heteroaryl” refers to an optionally substitutedmono- or bicyclic carbocyclic ring system having one or two aromaticrings containing from one to three heteroatoms and includes, but is notlimited to, furyl, thienyl, pyridyl and the like.

A “meroxapol” is polyoxypropylene-polyoxyethylene block copolymer withthe general formula HO(C₃H₆O)_(a)(C₂H₄O)_(b)(C3H₆O)_(a)H. It isavailable in different grades. Each meroxapol name is followed by a codenumber according to the average numerical values of the respectivemonomers units denoted by “a” and “b”.

As used herein, the term “optionally substituted” refers to from zero tofour substituents, wherein the substituents are each independentlyselected. Each of the independently selected substituents may be thesame or different than other substituents.

A “poloxamer” is a nonionic polyoxyethylene-polyoxypropylene blockcopolymer with the general formula HO(C₂H4O)_(a)(C3H₆O)b(C2H₄O)_(a)H. Itis available in different grades, which vary from liquids to solids.Each poloxamer name is followed by a code number according to theaverage numerical values of the respective monomers units denoted by “a”and “_(b)”.

A “poloxamine” is a polyoxyethylen-polyoxypropylene block copolymer ofethylene diamine with the general formula[HO(C₂H₄O)_(a)(C3H₆O)_(b)C3H₆]₂NCH₂CH₂N-[C3H₆(OC3H₆)_(b)(OC₂H4)_(a)OH]₂.It is available in different grades. Each poloxamine name is followed bya code number according to the average numerical values of therespective monomers units denoted by “a” and “b”.

The compounds of the present invention contain one or more asymmetriccenters in the molecule. In accordance with the present invention astructure that does not designate the stereochemistry is to beunderstood as embracing all the various optical isomers, as well asracemic mixtures thereof

The compounds of the present invention may exist in tautomeric forms andthe invention includes both mixtures and separate individual tautomers.For example the following structure:

is understood to represent a mixture of the structures:

The terminology used herein is for the purpose of describing theparticular versions or embodiments only, and is not intended to limitthe scope of the present invention. All publications mentioned hereinare incorporated by reference in their entirety.

In some embodiments, the present invention provides methods of using asurface active copolymer, including, but not limited to poloxamers, toincrease oxygenation, reduce bacteria, and/or improve healing oftissues. In some embodiments, the present invention provides methodscomprising the topical application of a composition or formulationcomprising at least one poloxamer or other surface active copolymer toan injured or diseased site to increase oxygenation, reduce bacteria,and/or improve healing at the site. The formulation may compriseadditional therapeutic agents, for example, antimicrobial agents toprevent infection, growth factors or hormones to enhance healing, drugsto treat inflammation, or anesthetics to decrease pain.

In some embodiments, the composition or formulation comprises apoloxamer (e.g. poloxamer-188 (Pluronic F68)). Not being bound by anytheory, a poloxamer has the special ability to thicken at highertemperatures (such as body temperature) and liquefy at coolertemperatures (cool rinse water for example). The thickness, orviscosity, varies depending on the amount or concentration of surfaceactive copolymer used. These properties enable it to remain resident attissue surfaces at body temperature but also enable it to be easilyremoved away with cool water. In some embodiments, the surface activecopolymer is biocompatible. In some embodiments, dilutions of surfaceactive copolymers such as Poloxamer-188 (Pluronic F68) arebiocompatible. In some embodiments, the poloxamer is present in acomposition or formulation in about 0.1, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10% w/w. In some embodiments, the poloxamer is present in acomposition or formulation in about 0.1-10% w/w.

Injuries, Wounds, Diseases, and Disorders

In some embodiments, the present invention provides methods of treatinga site of either healthy tissue or injured tissue. The types ofinjuries, disease, and disorders include, but are not limited to, burns,chronic wounds, and surgical procedures such as microvascular surgery,skin flaps and skin grafts, and tissue injury resulting from, forexample, a burn, scrape, cut, incision, laceration, ulcer, bodypiercing, bite wound, trauma, stab wound, gunshot wound, surgical wound,stretch injury, crush wound, compression wound, fracture, sprain,strain, stroke, infarction, aneurysm, herniation, ischemia, fistula,dislocation, radiation, cell, tissue or organ grafting, injuriessustained during medical procedures, or cancer.

Such injuries include, but are not limited to, skin injury, muscleinjury, brain injury, eye injury, or spinal cord injury. Tissue injurycan include joint injury, back injury, heart injury, vascular systeminjury, soft tissue injury, cartilage injury, lymphatic system injury,tendon injury, ligament injury, or abdominal injury.

The injuries that are contemplated to be treated by use of a compound,composition, or formulation as described herein, for example, anydenuded area without skin or mucosa that is due to trauma such as aburn, a surgical trauma, an abrasion, a malignancy, an infection, or anallergic reaction. It is believed that the use of the composition,compound, or formulation can result in an improved cosmetic andfunctional outcome for the subjects being treated.

The invention encompasses treatment of all types of thermal injuries andburns. These include acute conditions such as thermal burns, chemicalburns, radiation burns, burns caused by excess exposure to ultravioletradiation such as sunburn, as well as by the chronic wounds associatedwith some of these conditions.

Burns include first degree burns which may cause skin manifestationssuch as reddening, pain, and/or mild swelling. One non-limiting exampleof first degree burn is a sun burn. Burns further refers tosecond-degree burns involving the first two layers of skin. Signs ofsecond degree burning include, among other things, deep reddening of theskin, blisters, pain, glossy appearance from leaking fluid, and possibleskin loss. Burns further refers to third-degree burns which penetratethe entire thickness of the skin and may destroy tissue. Signs of thirddegree burning include, among other things, loss of skin, dry skin,leathery skin, charred skin having a mottled appearance, andcombinations thereof. In some cases, skin with a third degree burn maybe painless.

In some embodiments, compositions, compounds, or formulas disclosedherein will benefit, for example, subjects suffering from vesicant burnsand thermal burns, including first degree burns, second degree burns andthird degree burns, as well as esophageal burns and erosions. Forexample, after cutaneous burn injury, an area surrounding the wound isthe site of a pronounced inflammatory response. This “zone of stasis”undergoes progressive necrosis within 24-48 hours resulting in anexpansion of the burn wound.

Injuries encompassed by the invention further include acute and chronicwounds. Chronic wounds are wounds characterized by non-healing skinwounds and include chronic venous ulcers, diabetic ulcers, arterialulcers, pressure ulcers (e.g., decubitus ulcers), radiation ulcers,traumatic wounds, and open, complicated non-healing wounds. Woundsfurther refers to cuts and scrapes known as open wounds, as well asothers, such as deep bruises, or closed wounds. Non-limiting examples ofwounds suitable for treatment in accordance with the present disclosureinclude abrasions such as those caused by: scraping the outer layer ofskin; incisions such as those caused by sharp edges, knives, metaledges, broken glass or other sharp object; lacerations or jagged,irregular cuts or tears of the skin; punctures such as those caused byan object piercing the skin layers and creating a small hole; and/orburns. Additional non-limiting wounds suitable for treatment inaccordance with the present disclosure include puncture wounds, gapingwounds, wounds having fatty layers, tissue or muscle exposed, woundshaving one or more foreign bodies therein, wounds causing severe pain,wounds having blood flowing therefrom, or any wound that causes numbnessor loss of movement below the wound.

Other non-limiting examples of wounds suitable for treatment inaccordance with the present disclosure include animal bites, arterialdisease, insect stings and bites, bone infections, compromisedskin/muscle grafts, gangrene, skin tears or lacerations, surgicalincisions, including slow or non-healing surgical wounds, andpost-operation infections. It is understood, that the listed wounds arenon-limiting and that only a portion of wounds suitable for treatment inaccordance with the present disclosure are listed herein. It is alsocontemplated that the composition of the present invention will benefitsubjects with chronic skin ulcers, including but not limited todecubitus ulcers, venous stasis ulcers, arterial insufficiency ulcers,and diabetic foot ulcers.

Compositions and Formulations of the Base Surface Active Copolymer

The present invention provides for the preparation and use ofpharmaceutical compositions comprising as an active ingredient acompound useful for increasing oxygenation, reducing bacteria, and/orimproving healing at a tissue site. Such a pharmaceutical compositionmay consist of the active ingredient alone, in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise the active ingredient and one or more pharmaceuticallyacceptable carriers, one or more additional ingredients (e.g. additionaltherapeutic agent), or some combination of these. The active ingredientmay be present in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.The present invention further contemplates the use of more than oneactive ingredient.

The invention is not limited to the use of poloxamer-188 (Pluronic F68)but may include the use of a different or an additional surface activecopolymer, examples of which include other poloxamers, meroxapols, andpoloxamines. Examples of poloxamers include, but are not limited to,poloxamer-101, -105, -105 benzoate, -108, -122, -123, -124, -181, -182,-182 dibenzoate, -183, -184, -185, -188, -212, -215, -217, -231, -234,-235, -237, -238, -282, -284, -288, -331, -333, -334, -335, -338, -401,-402, -403, and -407. Examples of meroxapols include, but are notlimited to, meroxapol 105, 108, 171, 172, 174, 178, 251, 252, 254, 258,311, 312, and 314. Examples of poloxamines include, but are not limitedto, poloxamine 304, 504, 701, 702, 704, 707, 901, 904, 908, 1101, 1102,1104, 1301, 1302, 1304, 1307, 1501, 1502, 1504, and 1508.

In some embodiments, at least two different surface active copolymersare used. In one aspect, at least three different surface activecopolymers are used. These combinations may include, for example, one ormore poloxamers, one or more meroxapols, and one or more poloxamines.The copolymers of the invention may vary in size. The copolymer size mayrange, for example, from an M_(n) of about 600 to about 20,000, or inanother aspect from about 1,000 to about 10,000. In some embodiments,the weight of hydrophobic groups can be from about 45-95% by weight ofthe copolymer. A formulation comprising at least one copolymer (e.g., apoloxamer, meroxapol, or poloxamine) and water can be prepared bycooling it to an appropriate temperature, or by other methods known inthe art. Compositions of this type are described in U.S. Pat. No.5,635,540 (Edlich et al.), hereby incorporated by reference in itsentirety.

Examples of temperature ranges for preparation include, but are notlimited to, from about −20° C. to about 15° C., in another aspect fromabout −18° C. to about 8° C., and in another aspect, from about −15° C.to about 5° C. These ranges also encompass about 0° F. to about 60° F.One of ordinary skill in the art will understand that the temperaturesof preparation can be adjusted based on various criteria, such as thesurface active copolymer being used, the amount or concentration beingused, the type of formulation being prepared for administration, etc.

In some embodiments, the poloxamer base comprises 80% polyoxyethyleneunits and 20% polyoxypropylene units.

One of ordinary skill in the art will appreciate that the formulations,method of preparation, and amount of surface active copolymer used mayvary, depending on the type or location of the site to be treated. Forexample, in some embodiments, a poloxamer, such as poloxamer-188, ismixed with water at a ratio of from 1:0.8 to 1.2 w/w. This ratio can bevaried. This combination may be mixed until the powder has been wetted.The mixture may then be placed in a freezer or refrigerator and cooled,preferably for at least 4 hours. While cooling, the mixture will undergophase transition to a liquid, as demonstrated by Edlich et al. (U.S.Pat. No. 5,635,540). The mixture is then removed from the freezer andwarmed to room temperature. Pharmaceutical agents such as antimicrobialsand anesthetics can be added at this point, as demonstrated by Edlich etal. (U.S. Pat. No. 5,635,540). The poloxamer base used in preparing thetopical preparation can be a polyoxyalkylene based polymer based onethylene oxide and propylene oxide and comprises a series of closelyrelated block polymers that may generally be classified aspolyoxyethylene-polyoxypropylene condensates terminated in primaryhydroxyl groups. They are formed by the condensation of propylene oxideonto a propylene glycol nucleus followed by condensation of ethyleneoxide onto both ends of the polyoxypropylene base. The polyoxyethylenehydrophilic groups on the ends of the molecule are controlled in lengthto constitute anywhere from 10% to 90% by weight of the final molecule.

In some embodiments, the molecular weight Mn of the poloxamer baseranges from about 600 to about 20,000. In some embodiments, it rangesfrom about 1,000 to about 10,000. In some embodiments, it ranges fromabout 5,000 to about 8,500.

The compositions of the present invention may comprise one or moreco-additives (e.g., solvent such as water). In some embodiments, theconcentration of a surface active copolymer (e.g., poloxamer 188) isabout 0.01 to about 99.99% w/w. In some embodiments, it is about 1 toabout 90%. In some embodiments, it is about to about 80%. In someembodiments, it is about 20% to about 70%. In some embodiments, it isabout 50%. In some embodiments, it is about 5%. In some embodiments, theconcentration is a % w/w as disclosed herein.

In some embodiments, a formulation or composition can be impregnated ina dressing material (or otherwise contained or encompassed by thedressing material). The dressing material is a pharmaceuticallyacceptable fabric. It can be, for example, gauze or any other type ofmedical fabric or material that can be used to cover a wound and/or tokeep a therapeutic agent or composition in contact with a patient. Insome embodiments, the formulation or composition can be impregnated in awipe.

Additional Therapeutic Agents and Ingredients

The composition or formulation can further comprise additionaltherapeutic additives, alone or in combination (e.g., 2, 3, or 4additional additives). Examples of additional additives include but arenot limited to: (a) antimicrobials, (b) steroids (e.g., hydrocortisone,triamcinolone); (c) pain medications (e.g., aspirin, an NSAID, and alocal anesthetic); (d) anti-inflammatory agents; (e) growth factors; (f)cytokines; (g) hormones; and (h) combinations thereof.

In some embodiments, a composition or formulation comprises anantimicrobial agent. The antimicrobial agent may be provided at, forexample, a standard therapeutically effective amount. A standardtherapeutically effective amount is an amount that is typically used byone of ordinary skill in the art or an amount approved by a regulatoryagency (e.g., the FDA or its European counterpart). Antimicrobial agentsinclude those directed against the spectrums of gram positive organisms,gram negative organisms, fungi, and viruses. In some embodiments, thecomposition or formulation comprises a suitable local anesthetic agent.In some embodiments, a suitable local anesthetic agent has a meltingpoint of 30° to 70° C. Examples of suitable anesthetic agents include,but are not limited prilocaine, tetracaine, butanilcaine, trimecaine,benzocaine, lidocaine, bupivocaine, dibucaine, mepivocaine, andetidocaine.

In some embodiments, the methods use at least two anesthetics. The localanesthetic composition of the present invention may further comprisesuitable additives, such a pigment, a dye, an anti-oxidant, a stabilizeror a fragrance provided that addition of such an additive does notdestroy the single phase of the anesthetic composition.

In some embodiments, the hydrated local anesthetic mixture is preparedby melting the local anesthetic with the higher melting point of thetwo, followed by addition of the other local anesthetic, under vigorousmechanical mixing, such as trituration or grinding. A milky viscousliquid is formed, at which point, the surfactant is added with moremechanical mixing. Mixing of the surfactant produces a milky liquid ofsomewhat lower viscosity. Finally, the balance of water is added undervigorous mechanical mixing. The material can then be transferred to anair tight container, after which a clear composition is obtained afterabout 60 minutes at room temperature.

Alternatively, the hydrated local anesthetic mixture can be prepared byfirst melting the lower melting local anesthetic, followed by additionof the other local anesthetic along with vigorous mechanical mixing,then addition of the surfactant and water as above. However, when thelower melting local anesthetic is melted first, the storage time neededto obtain the single phase composition, increases from about 1 hour toabout 72 hours.

One of ordinary skill in the art will appreciate that there are multiplesuitable surfactants useful for preparing the hydrated topicalanesthetic of the present invention. For example, single-phase hydratedtopical anesthetics can be prepared from anionic, cationic or non-ionicsurfactants.

In some embodiments, the methods comprise using a composition comprisingany therapeutic molecule including, without limitation, anypharmaceutical or drug. Examples of pharmaceuticals include, but are notlimited to, anesthetics, hypnotics, sedatives and sleep inducers,antipsychotics, antidepressants, antiallergics, antianginals,antiarthritics, antiasthmatics, antidiabetics, antidiarrheal drugs,anticonvulsants, antigout drugs, antihistamines, antipruritics, emetics,antiemetics, antispasmodics, appetite suppressants, neuroactivesubstances, neurotransmitter agonists, antagonists, receptor blockersand reuptake modulators, beta-adrenergic blockers, calcium channelblockers, disulfiram and disulfiram-like drugs, muscle relaxants,analgesics, antipyretics, stimulants, anticholinesterase agents,parasympathomimetic agents, hormones, anticoagulants, antithrombotics,thrombolytics, immunoglobulins, immunosuppressants, hormoneagonists/antagonists, vitamins, antimicrobial agents, antineoplastics,antacids, digestants, laxatives, cathartics, antiseptics, diuretics,disinfectants, fungicides, ectoparasiticides, antiparasitics, heavymetals, heavy metal antagonists, chelating agents, gases and vapors,alkaloids, salts, ions, autacoids, digitalis, cardiac glycosides,antiarrhythmics, antihypertensives, vasodilators, vasoconstrictors,antimuscarinics, ganglionic stimulating agents, ganglionic blockingagents, neuromuscular blocking agents, adrenergic nerve inhibitors,anti-oxidants, vitamins, cosmetics, anti-inflammatories, wound careproducts, antithrombogenic agents, antitumoral agents, antiangiogenicagents, anesthetics, antigenic agents, wound healing agents, plantextracts, growth factors, emollients, humectants,rejection/anti-rejection drugs, spermicides, conditioners, antibacterialagents, antifungal agents, antiviral agents, antibiotics, tranquilizers,cholesterol-reducing drugs, antitussives, histamine-blocking drugs,monoamine oxidase inhibitor. All substances listed by the U.S.Pharmacopeia are also included within the substances of the presentinvention.

A list of the types of drugs, and specific drugs within categories isprovided below and are intended be non-limiting examples.

Antimicrobial agents include: silver sulfadiazine, Nystatin,Nystatin/triamcinolone, Bacitracin, nitrofurazone, nitrofurantoin, apolymyxin (e.g., Colistin, Surfactin, Polymyxin E, and Polymyxin B),doxycycline, antimicrobial peptides (e.g., natural and syntheticorigin), Neosporin (i.e., Bacitracin, Polymyxin B, and Neomycin),Polysporin (i.e., Bacitracin and Polymyxin B). Additional antimicrobialsinclude topical antimicrobials (i.e., antiseptics), examples of whichinclude silver salts, iodine, benzalkonium chloride, alcohol, hydrogenperoxide, and chlorhexidine.

Analgesic: Acetaminophen; Alfentanil Hydrochloride; AminobenzoatePotassium; Aminobenzoate Sodium; Anidoxime; Anileridine; AnileridineHydrochloride; Anilopam Hydrochloride; Anirolac; Antipyrine; Aspirin;Benoxaprofen; Benzydamine Hydrochloride; Bicifadine Hydrochloride;Brifentanil Hydrochloride; Bromadoline Maleate; Bromfenac Sodium;Buprenorphine Hydrochloride; Butacetin; Butixirate; Butorphanol;Butorphanol Tartrate; Carbamazepine; Carbaspirin Calcium; CarbipheneHydrochloride; Carfentanil Citrate; Ciprefadol Succinate; Ciramadol;Ciramadol Hydrochloride; Clonixeril; Clonixin; Codeine; CodeinePhosphate; Codeine Sulfate; Conorphone Hydrochloride; Cyclazocine;Dexoxadrol Hydrochloride; Dexpemedolac; Dezocine; Diflunisal;Dihydrocodeine Bitartrate; Dimefadane; Dipyrone; DoxpicomineHydrochloride; Drinidene; Enadoline Hydrochloride; Epirizole; ErgotamineTartrate; Ethoxazene Hydrochloride; Etofenamate; Eugenol; Fenoprofen;Fenoprofen Calcium; Fentanyl Citrate; Floctafenine; Flufenisal;Flunixin; Flunixin Meglumine; Flupirtine Maleate; Fluproquazone;Fluradoline Hydrochloride; Flurbiprofen; Hydromorphone Hydrochloride;Ibufenac; Indoprofen; Ketazocine; Ketorfanol; Ketorolac Tromethamine;Letimide Hydrochloride; Levomethadyl Acetate; Levomethadyl AcetateHydrochloride; Levonantradol Hydrochloride; Levorphanol Tartrate;Lofemizole Hydrochloride; Lofentanil Oxalate; Lorcinadol; Lomoxicam;Magnesium Salicylate; Mefenamic Acid; Menabitan Hydrochloride;Meperidine Hydrochloride; Meptazinol Hydrochloride; MethadoneHydrochloride; Methadyl Acetate; Methopholine; Methotrimeprazine;Metkephamid Acetate; Mimbane Hydrochloride; Mirfentanil Hydrochloride;Molinazone; Morphine Sulfate; Moxazocine; Nabitan Hydrochloride;Nalbuphine Hydrochloride; Nalmexone Hydrochloride; Namoxyrate; NantradolHydrochloride; Naproxen; Naproxen Sodium; Naproxol; NefopamHydrochloride; Nexeridine Hydrochloride; Noracymethadol Hydrochloride;Ocfentanil Hydrochloride; Octazamide; Olvanil; Oxetorone Fumarate;Oxycodone; Oxycodone Hydrochloride; Oxycodone Terephthalate; OxymorphoneHydrochloride; Pemedolac; Pentamorphone; Pentazocine; PentazocineHydrochloride; Pentazocine Lactate; Phenazopyridine Hydrochloride;Phenyramidol Hydrochloride; Picenadol Hydrochloride; Pinadoline;Pirfenidone; Piroxicam Olamine; Pravadoline Maleate; ProdilidineHydrochloride; Profadol Hydrochloride; Propirarn Fumarate; PropoxypheneHydrochloride; Propoxyphene Napsylate; Proxazole; Proxazole Citrate;Proxorphan Tartrate; Pyrroliphene Hydrochloride; RemifentanilHydrochloride; Salcolex; Salethamide Maleate; Salicylamide; SalicylateMeglumine; Salsalate; Sodium Salicylate; Spiradoline Mesylate;Sufentanil; Sufentanil Citrate; Talmetacin; Talniflumate; Talosalate;Tazadolene Succinate; Tebufelone; Tetrydamine; Tifurac Sodium; TilidineHydrochloride; Tiopinac; Tonazocine Mesylate; Tramadol Hydrochloride;Trefentanil Hydrochloride; Trolamine; Veradoline Hydrochloride;Verilopam Hydrochloride; Volazocine; Xorphanol Mesylate; XylazineHydrochloride; Zenazocine Mesylate; Zomepirac Sodium; Zucapsaicin.

Antihypertensive: Aflyzosin Hydrochloride; Alipamide; Althiazide;Amiquinsin Hydrochloride; Amlodipine Besylate; Amlodipine Maleate;Anaritide Acetate; Atiprosin Maleate; Belfosdil; Bemitradine; BendacalolMesylate; Bendroflumethiazide; Benzthiazide; Betaxolol Hydrochloride;Bethanidine Sulfate; Bevantolol Hydrochloride; Biclodil Hydrochloride;Bisoprolol; Bisoprolol Fumarate; Bucindolol Hydrochloride; Bupicomide;Buthiazide: Candoxatril; Candoxatrilat; Captopril; Carvedilol;Ceronapril; Chlorothiazide Sodium; Cicletanine; Cilazapril; Clonidine;Clonidine Hydrochloride; Clopamide; Cyclopenthiazide; Cyclothiazide;Darodipine; Debrisoquin Sulfate; Delapril Hydrochloride; Diapamide;Diazoxide; Dilevalol Hydrochloride; Diltiazem Malate; Ditekiren;Doxazosin Mesylate; Ecadotril; Enalapril Maleate; Enalaprilat;Enalkiren; Endralazine Mesylate; Epithiazide; Eprosartan; EprosartanMesylate; Fenoldopam Mesylate; Flavodilol Maleate; Flordipine;Flosequinan; Fosinopril Sodium; Fosinoprilat; Guanabenz; GuanabenzAcetate; Guanacline Sulfate; Guanadrel Sulfate; Guancydine; GuanethidineMonosulfate; Guanethidine Sulfate; Guanfacine Hydrochloride; GuanisoquinSulfate; Guanoclor Sulfate; Guanoctine Hydrochloride; Guanoxabenz;Guanoxan Sulfate; Guanoxyfen Sulfate; Hydralazine Hydrochloride;Hydralazine Polistirex; Hydroflumethiazide; Indacrinone; Indapamide;Indolaprif Hydrochloride; Indoramin; Indoramin Hydrochloride; IndorenateHydrochloride; Lacidipine; Leniquinsin; Levcromakalim; Lisinopril;Lofexidine Hydrochloride; Losartan Potassium; Losulazine Hydrochloride;Mebutamate; Mecamylamine Hydrochloride; Medroxalol; MedroxalolHydrochloride; Methalthiazide; Methyclothiazide; Methyldopa;Methyldopate Hydrochloride; Metipranolol; Metolazone; MetoprololFumarate; Metoprolol Succinate; Metyrosine; Minoxidil; MonatepilMaleate; Muzolimine; Nebivolol; Nitrendipine; Ofornine; PargylineHydrochloride; Pazoxide; Pelanserin Hydrochloride; Perindopril Erbumine;Phenoxybenzamine Hydrochloride; Pinacidil; Pivopril; Polythiazide;Prazosin Hydrochloride; Primidolol; Prizidilol Hydrochloride; QuinaprilHydrochloride; Quinaprilat; Quinazosin Hydrochloride; QuineloraneHydrochloride; Quinpirole Hydrochloride; Quinuclium Bromide; Ramipril;Rauwolfia Serpentina; Reserpine; Saprisartan Potassium; SaralasinAcetate; Sodium Nitroprusside; Sulfinalol Hydrochloride; Tasosartan;Teludipine Hydrochloride; Temocapril Hydrochloride; TerazosinHydrochloride; Terlakiren; Tiamenidine; Tiamenidine Hydrochloride;Ticrynafen; Tinabinol; Tiodazosin; Tipentosin Hydrochloride;Trichlormethiazide; Trimazosin Hydrochloride; Trimethaphan Camsylate;Trimoxamine Hydrochloride; Tripamide; Xipamide; Zankiren Hydrochloride;Zofenoprilat Arginine.

Anti-inflammatory: Alclofenac; Alclometasone Dipropionate; AlgestoneAcetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium;Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone;Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac;Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort;Desonide; Desoximetasone; Dexamethasone Dipropionate; DiclofenacPotassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Momiflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Zomepirac Sodium.

Growth Factors

In some embodiments, an effective amount of at least one growth factor,cytokine, hormone, or extracellular matrix compound or protein usefulfor enhancing wound healing is administered. In some embodiments, acombination of these agents is used. In some embodiments, examples ofgrowth factors include, but are not limited to, EGF, PDGF, GCSF, IL6,IL8, IL10, MCP1, MCP2, Tissue Factor, FGFb, KGF, VEGF, PLGF, MMPI, MMP9,TIMP1, TIMP2, TGFβ, and HGF. One of ordinary skill in the art willappreciate that the choice of growth factor, cytokine, hormone, orextracellular matrix protein used will vary depending on criteria suchas the type of injury, disease, or disorder being treated, the age,health, sex, and weight of the subject, etc. In some embodiments, thegrowth factors, cytokines, hormones, and extracellular matrix compoundsand proteins are human.

Proteins and other biologically active compounds that can beincorporated into, or included as an additive within a composition orformulation described herein include, but are not limited to, collagen(including cross-linked collagen), fibronectin, laminin, elastin(including cross-linked elastin), osteopontin, osteonectin, bonesialoproteins (Bsp), alpha-2HS-glycoproteins, bone Gla-protein (Bgp),matrix Gla-protein, bone phosphoglycoprotein, bone phosphoprotein, boneproteoglycan, protolipids, bone morphogenetic protein, cartilageinduction factor, skeletal growth factor, enzymes, or combinations andbiologically active fragments thereof. Adjuvants that diminish an immuneresponse can also be used in conjunction with the composite of thesubject invention. Other molecules that can be used include, but are notlimited to, growth hormones, leptin, leukemia inhibitory factor (LIF),tumor necrosis factor alpha and beta, endostatin, angiostatin,thrombospondin, osteogenic protein-1, bone morphogenetic proteins 2 and7, osteonectin, somatomedin-like peptide, osteocalcin, interferon alpha,interferon alpha A, interferon beta, interferon gamma, interferon 1alpha, and interleukins 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12, 13, 15, 16,17 and 18. Embodiments involving amino acids, peptides, polypeptides,and proteins may include any type of such molecules of any size andcomplexity as well as combinations of such molecules.

Pharmaceutical Compositions and Delivery Form

The compositions and formulations may be prepared in a variety of formsknown in the art, such as liquids, aerosols, or gels. Topicaladministration can be performed by, for example, hand, mechanically(e.g., extrusion and spray delivery) or as a component of a dressing(e.g., gauze or other wound covering). The administration of thecomposition or formulation directly by hand to a tissue or biomaterialsurface is preformed so as to achieve a therapeutic coating, which maybe uniform, alone or in combination with an overlying dressing.

In some embodiments, the administration of the composition orformulation mechanically can be performed by using a device thatphysically pushes the composition onto a tissue or biomaterial surfaceso as to achieve a therapeutic coating, which may be uniform, alone orin combination with an overlying dressing.

In some embodiments, the composition or formulation can be sprayed ontoa tissue or biomaterial surface so as to achieve a therapeutic coating,which may be uniform, alone or in combination with an overlyingdressing. When part of a dressing, the composition or formulation isapplied so as to achieve a therapeutic coating of the surface, which maybe uniform.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 70% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

In some embodiments, a pharmaceutical cream is provided wherein apoloxamer base, in the form of powder, is mixed with water, and causedto become hydrated, by subjecting the combination of poloxamer base andwater, to freezing temperatures, before an additional pharmaceuticalagent such as an additional therapeutic agent is added.

In some embodiments, the present invention provides for the preparationand use of pharmaceutical compositions comprising a compound useful fortreatment of various skin related injuries, trauma, diseases, disorders,or conditions described herein, including burns, wounds, surgicalincisions, etc. The invention also encompasses other injuries, trauma,associated diseases and disorders other than those of the skin,including, but not limited to, gum diseases and disorders. Such apharmaceutical composition may consist of the active ingredient alone,in a form suitable for administration to a subject, or thepharmaceutical composition may comprise at least one active ingredientand one or more pharmaceutically acceptable carriers, one or moreadditional ingredients, or some combination of these. The activeingredient may be present in the pharmaceutical composition in the formof a physiologically acceptable ester or salt, such as in combinationwith a physiologically acceptable cation or anion, as is well known inthe art.

An obstacle for topical administration of pharmaceuticals to the skin isthe stratum corneum layer of the epidermis. The stratum corneum is ahighly resistant layer comprised of protein, cholesterol, sphingolipids,free fatty acids and various other lipids, and includes cornified andliving cells. One of the factors that limits the penetration rate (flux)of a compound through the stratum corneum is the amount of the activesubstance which can be loaded or applied onto the skin surface. Thegreater the amount of active substance which is applied per unit of areaof the skin, the greater the concentration gradient between the skinsurface and the lower layers of the skin, and in turn the greater thediffusion force of the active substance through the skin. Therefore, aformulation containing a greater concentration of the active substanceis more likely to result in penetration of the active substance throughthe skin, and more of it, and at a more consistent rate, than aformulation having a lesser concentration, all other things being equal.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

The compositions or formulations described herein may be administeredto, for example, a cell, a tissue, or a subject by any of severalmethods described herein and by others which are known to those of skillin the art.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,sex, age, size, and condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active or therapeutic agents. In some embodiments,additional agents include anti-emetics and scavengers such as cyanideand cyanate scavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Additionally, formulations for topical administration may includeliquids, ointments, lotions, creams, gels (e.g., poloxamer gel), drops,suppositories, sprays, aerosols, and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. The disclosed compositions canbe administered, for example, in a microfiber, polymer (e.g., collagen),nanosphere, aerosol, lotion, cream, fabric, plastic, tissue engineeredscaffold, matrix material, tablet, implanted container, powder, oil,resin, wound dressing, bead, microbead, slow release bead, capsule,injectables, intravenous drips, pump device, silicone implants, or anybio-engineered materials.

Enhancers of permeation may be used. These materials increase the rateof penetration of drugs across the skin. Typical enhancers in the artinclude ethanol, glycerol monolaurate, PGML (polyethylene glycolmonolaurate), dimethylsulfoxide, and the like. Other enhancers includeoleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylicacids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositionsof the invention may contain liposomes. The composition of the liposomesand their use are known in the art (for example, see Constanza, U.S.Pat. No. 6,323,219).

The source of active compound to be formulated will generally dependupon the particular form of the compound. Small organic molecules andpeptidyl or oligo fragments can be chemically synthesized and providedin a pure form suitable for pharmaceutical/cosmetic usage. Products ofnatural extracts can be purified according to techniques known in theart. Recombinant sources of compounds are also available to those ofordinary skill in the art.

In some embodiments, the topically active pharmaceutical composition maybe optionally combined with other ingredients such as moisturizers,cosmetic adjuvants, anti-oxidants, chelating agents, bleaching agents,tyrosinase inhibitors, and other known depigmentation agents,surfactants, foaming agents, conditioners, humectants, wetting agents,emulsifying agents, fragrances, viscosifiers, buffering agents,preservatives, sunscreens, and the like. In some embodiments, apermeation or penetration enhancer is included in the composition and iseffective in improving the percutaneous penetration of the activeingredient into and through the stratum corneum with respect to acomposition lacking the permeation enhancer. Various permeationenhancers, including oleic acid, oleyl alcohol, ethoxydiglycol,laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, orN-methyl-2-pyrrolidone, are known to those of skill in the art. In someembodiments, the composition may further comprise a hydrotropic agent,which functions to increase disorder in the structure of the stratumcorneum, and thus allows increased transport across the stratum corneum.Various hydrotropic agents such as isopropyl alcohol, propylene glycol,or sodium xylene sulfonate, are known to those of skill in the art. Thecompositions may also contain active amounts of retinoids (i.e.,compounds that bind to any members of the family of retinoid receptors),including, for example, tretinoin, retinol, esters of tretinoin and/orretinol and the like.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts.

The present invention encompasses biologically active analogs, homologs,derivatives, and modifications of the compounds of the invention.Methods for the preparation of such compounds are known in the art.

Modification of pharmaceutical compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and perform suchmodification with merely ordinary, if any, experimentation.

The composition and formulations, including but not limited to,pharmaceutical compositions, can be administered according to themethods described herein to any animal, such as a mammal (e.g. human,primates and non-primates). In some embodiments, subjects to whichadministration of the pharmaceutical compositions of the invention iscontemplated include, but are not limited to, humans and other primates,mammals including commercially relevant mammals such as cattle, pigs,horses, sheep, cats, and dogs.

Liquid derivatives and natural extracts made directly from biologicalsources may be employed in the compositions of this invention in aconcentration (w/w) from about 1 to about 99%. Fractions of naturalextracts and protease inhibitors may have a different preferred rage,from about 0.01% to about 20% and, more preferably, from about 1% toabout 10% of the composition. Of course, mixtures of the active agentsof this invention may be combined and used together in the sameformulation, or in serial applications of different formulations.

The composition may comprise a preservative from about 0.005% to 2.0% bytotal weight of the composition. The preservative is used to preventspoilage in the case of an aqueous gel because of repeated patient usewhen it is exposed to contaminants in the environment from, for example,exposure to air or the patient's skin, including contact with thefingers used for applying a composition of the invention such as atherapeutic gel or cream. Examples of preservatives useful in accordancewith the invention included but are not limited to those selected fromthe group consisting of benzyl alcohol, sorbic acid, parabens, imidurea,and combinations thereof. A particularly preferred preservative is acombination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5%sorbic acid. The composition may include an antioxidant and a chelatingagent which inhibit the degradation of the compound for use in theinvention in the aqueous gel formulation. Preferred antioxidants forsome compounds are BHT, BHA, alphatocopherol, and ascorbic acid in thepreferred range of about 0.01% to 0.3% and more preferably BHT in therange of 0.03% to 0.1% by weight by total weight of the composition.Preferably, the chelating agent is present in an amount of from 0.01% to0.5% by weight by total weight of the composition. Particularlypreferred chelating agents include edetate salts (e.g. disodium edetate)and citric acid in the weight range of about 0.01% to 0.20% and morepreferably in the range of 0.02% to 0.10% by weight by total weight ofthe composition. The chelating agent is useful for chelating metal ionsin the composition which may be detrimental to the shelf life of theformulation. While BHT and disodium edetate are preferred antioxidantand chelating agent respectively for some compounds, other suitable andequivalent antioxidants and chelating agents may be substituted thereforas would be known to those skilled in the art.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

Other components such as preservatives, antioxidants, surfactants,absorption enhancers, viscosity enhancers or film forming polymers,bulking agents, diluents, coloring agents, flavoring agents, pHmodifiers, sweeteners or taste-masking agents may also be incorporatedinto the composition. Suitable coloring agents include red, black, andyellow iron oxides and FD&C dyes such as FD&C Blue No. 2, FD&C Red No.40, and the like. Suitable flavoring agents include mint, raspberry,licorice, orange, lemon, grapefruit, caramel, vanilla, cherry grapeflavors, combinations thereof, and the like. Suitable pH modifiersinclude citric acid, tartaric acid, phosphoric acid, hydrochloric acid,maleic acid, sodium hydroxide, and the like. Suitable sweeteners includeaspartame, acesulfame K, thaumatic, and the like. Suitable taste-maskingagents include sodium bicarbonate, ion-exchange resins, cyclodextrininclusion compounds, adsorbates, and the like.

Absorption enhancers for use in accordance with the present inventioninclude, for example, polysorbates, sorbitan esters, poloxamer blockcopolymers, PEG-35 castor oil, PEG-40 hydrogenated castor oil,caprylocaproyl macrogol-8 glycerides, PEG-8 caprylic/capric glycerides,sodium lauryl sulfate, dioctyl sulfosuccinate, polyethylene laurylether, ethoxydiglycol, propylene glycol mono-di-caprylate, glycerolmonocaprylate, glyceryl fatty acids, oleic acid, linoleic acid, glycerylcaprylate/caprate, glyceryl monooleate, glyceryl monolaurate,caprylic/capric triglycerides, ethoxylated nonylphenols, PEG-(8-50)stearates, olive oil PEG-6 esters, triolein PEG-6 esters, lecithin,d-alpha tocopheryl polyethylene glycol 1000 succinate, polycarbonate,sodium glycocholate, sodium taurocholate, cyclodextrins, citric acid,sodium citrate, triacetin, combinations thereof, and the like. In someembodiments, the absorption enhancer is triacetin. In some embodimentswhere an absorption enhancer is included in the formulation, theabsorption enhancer is included in an amount of from about 0.001% toabout 10% by weight of the formulation, preferably in an amount of about0.01% to about 5% by weight of the formulation.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Modification of pharmaceutical compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and perform suchmodification with merely ordinary, if any, experimentation. Subjects towhich administration of the pharmaceutical compositions of the inventionis contemplated include, but are not limited to, humans and otherprimates, mammals including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, and dogs, and birds includingcommercially relevant birds such as chickens, ducks, geese, and turkeys.

The pharmaceutical compositions of the invention can be administered inany suitable formulation, by any suitable means, and by any suitableroute of administration. Formulations suitable for topicaladministration include, but are not limited to, liquid or semi-liquidpreparations such as liniments, lotions, oil in water or water in oilemulsions such as creams, ointments or pastes, and solutions orsuspensions. Topically-administrable formulations may, for example,comprise from about 1% to about 10% (w/w) active ingredient, althoughthe concentration of the active ingredient may be as high as thesolubility limit of the active ingredient in the solvent. Formulationsfor topical administration may further comprise one or more of theadditional ingredients described herein.

Topical administration of compositions of the invention may includetransdermal application. Transdermal application can be performed eitherpassively or using iontophoresis or electroporation.

Compositions of the invention may be applied using transdermal patches.Transdermal patches are adhesive backed patches laced with an effectiveamount of compounds of the invention. The pressure-sensitive adhesive ofthe matrix will normally be a solution of polyacrylate, a silicone, orpolyisobutylene (PIB). Such adhesives are well known in the transdermalart. See, for instance, the Handbook of Pressure Sensitive AdhesiveTechnology, 2nd Edition (1989) Van Nostrand, Reinhold.

Pressure sensitive solution polyacrylate adhesives for transdermalpatches are made by copolymerizing one or more acrylate monomers(“acrylate” is intended to include both acrylates and methacrylates),one or more modifying monomers, and one or more functionalgroup-containing monomers in an organic solvent. The acrylate monomersused to make these polymers are normally alkyl acrylates of 4-17 carbonatoms, with 2-ethylhexyl acrylate, butyl acrylate, and isooctyl acrylatebeing preferred. Modifying monomers are typically included to alter theTg of the polymer. Such monomers as vinyl acetate, ethyl acrylate andmethacrylate, and methyl methacrylate are useful for this purpose. Thefunctional group-containing monomer provides sites for crosslinking. Thefunctional groups of these monomers are preferably carboxyl, hydroxy orcombinations thereof Examples of monomers that provide such groups areacrylic acid, methacrylic acid and hydroxy-containing monomers such ashydroxyethyl acrylate. The polyacrylate adhesives are preferablycrosslinked using a crosslinking agent to improve their physicalproperties, (e.g., creep and shear resistance). The crosslinking densityshould be low since high degrees of crosslinking may affect the adhesiveproperties of the copolymer adversely. Examples of crosslinking agentsare disclosed in U.S. Pat. No. 5,393,529. Solution polyacrylate pressuresensitive adhesives are commercially available under tradenames such asGELVA™ and DURO-TAK™ from 3M.

Polyisobutylene adhesives are mixtures of high molecular weight (HMW)PIB and low molecular weight (LMW) PIB. Such mixtures are described inthe art, e.g., PCT/US91/02516. The molecular weight of the HMW PIB willusually be in the range of about 700,000 to 2,000,000 Da, whereas thatof the LMW PIB will typically range between 35,000 to 60,000. Themolecular weights referred to herein are weight average molecularweight. The weight ratio of HMW PIB to LMW PIB in the adhesive willnormally range between 1:1 to 1:10. The PIB adhesive will also normallyinclude a tackifier such as polybutene oil and high Tg, low molecularweight aliphatic resins such as the ESCOREZ™ resins available from ExxonChemical. Polyisobutylene polymers are available commercially under thetradename VISTANEX™ from Exxon Chemical.

The silicone adhesives that may be used in forming the matrix aretypically high molecular weight poly dimethyl siloxanes orpolydimethyldiphenyl siloxanes. Formulations of silicone adhesives thatare useful in transdermal patches are described in U.S. Pat. Nos.5,232,702, 4,906,169, and 4,951,622.

Dosage forms for topical or transdermal administration of a compound ofthis invention include liquids, ointments, pastes, creams, lotions,gels, powders, solutions, sprays, aerosols, inhalants or patches. Theactive component is admixed under sterile conditions with apharmaceutically acceptable carrier and any needed preservatives orbuffers as may be required. Ophthalmic formulation, ear drops, eyeointments, powders, and solutions are also contemplated as being withinthe scope of this invention. Transdermal patches have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms can be made by dissolving or dispensing thecompound(s) in the proper medium. Absorption enhancers can also be usedto increase the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The ointments, pastes, creams, and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof

Topical administration may also be performed using iontophoresisdevices. Such delivery systems eliminate needles entirely, and rely uponchemical mediators or external driving forces such as iontophoreticcurrents or thermal poration or sonophoresis to breach the stratumcorneum, the outermost layer of the skin, and deliver substances throughthe surface of the skin. The process of iontophoresis has foundcommercial use in the delivery of ionically charged therapeutic agentmolecules such as pilocarpine, lidocaine, and dexamethasone. In thisdelivery method, ions bearing a positive charge are driven across theskin at the site of an electrolytic electrical system anode while ionsbearing a negative charge are driven across the skin at the site of anelectrolytic system cathode.

The present invention provides a system for the direct application ofcompounds of the invention, including additional therapeutic agents suchas anesthetic agents, by iontophoresis for increasing oxygenation,reducing bacteria, and/or improving healing at a site while decreasingpain associated with injuries, diseases, and disorders.

In one embodiment, the methods of the invention provide a patch devicewith a donor or delivery chamber that is designed to be applied directlyover an injury, incision, or wound site and utilizes an electric fieldto stimulate delivery of the active compound or additional therapeuticagents(s). The patch is sterilized so that risk of infection is minimal.Additionally, the system delivers medication in a constant manner overan extended period of time. Generally, such time periods are at least 30minutes and may extend to as many as 96 hours. A pharmaceuticalcomposition may be prepared, packaged, or sold in a formulation suitablefor pulmonary administration via the buccal cavity. Such a formulationmay comprise dry particles which comprise the active ingredient andwhich have a diameter in the range from about 0.5 to about 7 nanometers,and preferably from about 1 to about 6 nanometers. Such compositions areconveniently in the form of dry powders for administration using adevice comprising a dry powder reservoir to which a stream of propellantmay be directed to disperse the powder or using a self-propellingsolvent/powder-dispensing container such as a device comprising theactive ingredient dissolved or suspended in a low-boiling propellant ina sealed container. In some embodiments, such powders comprise particleswherein at least 98% of the particles by weight have a diameter greaterthan 0.5 nanometers and at least 95% of the particles by number have adiameter less than 7 nanometers. In some embodiments, at least 95% ofthe particles by weight have a diameter greater than 1 nanometer and atleast 90% of the particles by number have a diameter less than 6nanometers. Dry powder compositions, in some embodiments, include asolid fine powder diluent such as sugar and are conveniently provided ina unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally, thepropellant may constitute about 50% to about 99.9% (w/w) of thecomposition, and the active ingredient may constitute about 0.1% toabout 20% (w/w) of the composition. The propellant may further compriseadditional ingredients such as a liquid non-ionic or solid anionicsurfactant or a solid diluent (preferably having a particle size of thesame order as particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration can have an average diameter inthe range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to about 500 micrometers. Such a formulation isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as about 0.1% (w/w) and as much as about100% (w/w) of the active ingredient, and may further comprise one ormore of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, comprise about0.1% to about 20% (w/w) active ingredient, the balance comprising anorally dissolvable or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powder oran aerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or atomized formulations, whendispersed, preferably have an average particle or droplet size in therange from about 0.1 to about 200 nanometers, and may further compriseone or more of the additional ingredients described herein.Additionally, the formulation taken orally can be prepared as apharmaceutical composition, including, but not limited to, a paste, agel, a toothpaste, a mouthwash, a solution, an oral rinse, a suspension,an ointment, a cream, and a coating.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1% to 1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for intramucosal administration. Thepresent invention provides for intramucosal administration of compoundsto allow passage or absorption of the compounds across mucosa. Such typeof administration is useful for absorption orally (gingival, sublingual,buccal, etc.), rectally, vaginally, pulmonary, nasally, etc.

The compounds of the invention can be prepared in a formulation orpharmaceutical composition appropriate for administration that allows orenhances absorption across mucosa. Mucosal absorption enhancers include,but are not limited to, a bile salt, fatty acid, surfactant, or alcohol.In some embodiments, the permeation enhancer can be sodium cholate,sodium dodecyl sulphate, sodium deoxycholate, taurodeoxycholate, sodiumglycocholate, dimethylsulfoxide, or ethanol. In some embodiments, acompound of the invention can be formulated with a mucosal penetrationenhancer to facilitate delivery of the compound. The formulation canalso be prepared with pH optimized for solubility, drug stability, andabsorption through mucosa such as nasal mucosa, oral mucosa, vaginalmucosa, respiratory, and intestinal mucosa.

To further enhance mucosal delivery of pharmaceutical agents,formulations comprising the active agent may also contain a hydrophiliclow molecular weight compound as a base or excipient. Such hydrophiliclow molecular weight compounds provide a passage medium through which awater-soluble active agent, such as a physiologically active peptide orprotein, may diffuse through the base to the body surface where theactive agent is absorbed. The hydrophilic low molecular weight compoundoptionally absorbs moisture from the mucosa or the administrationatmosphere and dissolves the water-soluble active peptide. The molecularweight of the hydrophilic low molecular weight compound is generally notmore than 10000 and preferably not more than 3000. Exemplary hydrophiliclow molecular weight compounds include polyol compounds, such as oligo-,di- and monosaccharides such as sucrose, mannitol, lactose, L-arabinose,D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose,cellobiose, gentibiose, glycerin, and polyethylene glycol. Otherexamples of hydrophilic low molecular weight compounds useful ascarriers within the invention include N-methylpyrrolidone, and alcohols(e.g., oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol,etc.). These hydrophilic low molecular weight compounds can be usedalone or in combination with one another or with other active orinactive components of the intranasal formulation.

When a controlled-release pharmaceutical preparation further contains ahydrophilic base, many options are available for inclusion. Hydrophilicpolymers such as a polyethylene glycol and poly vinyl pyrrolidone, sugaralcohols such as D-sorbitol and xylitol, saccharides such as sucrose,maltose, lactulose, D-fructose, dextran, and glucose, surfactants suchas polyoxyethylene-hydrogenated castor oil, polyoxyethylenepolyoxypropylene glycol, and polyoxyethylene sorbitan higher fatty acidesters, salts such as sodium chloride and magnesium chloride, organicacids such as citric acid and tartaric acid, amino acids such asglycine, beta-alanine, and lysine hydrochloride, and aminosaccharidessuch as meglumine are given as examples of the hydrophilic base.Polyethylene glycol, sucrose, and polyvinyl pyrrolidone are preferredand polyethylene glycol are further preferred. One or a combination oftwo or more hydrophilic bases can be used.

The present invention contemplates pulmonary, nasal, or oraladministration through an inhaler. In some embodiments, delivery from aninhaler can be a metered dose.

An inhaler is a device for patient self-administration of at least onecompound of the invention comprising a spray inhaler (e.g., a nasal,oral, or pulmonary spray inhaler) containing an aerosol sprayformulation of at least one compound of the invention and apharmaceutically acceptable dispersant. In some embodiments, the deviceis metered to disperse an amount of the aerosol formulation by forming aspray that contains a dose of at least one compound of the inventioneffective to treat a disease or disorder encompassed by the invention.The dispersant may be a surfactant, such as, but not limited to,polyoxyethylene fatty acid esters, polyoxyethylene fatty acid alcohols,and polyoxyethylene sorbitan fatty acid esters. Phospholipid-basedsurfactants also may be used.

In some embodiments, the aerosol formulation is provided as a dry powderaerosol formulation in which a compound of the invention is present as afinely divided powder. The dry powder formulation can further comprise abulking agent, such as, but not limited to, lactose, sorbitol, sucrose,and mannitol.

In some embodiments, the aerosol formulation is a liquid aerosolformulation further comprising a pharmaceutically acceptable diluent,such as, but not limited to, sterile water, saline, buffered saline anddextrose solution.

In some embodiments, the aerosol formulation further comprises at leastone additional compound of the invention in a concentration such thatthe metered amount of the aerosol formulation dispersed by the devicecontains a dose of the additional compound in a metered amount that iseffective to ameliorate the symptoms of disease or disorder disclosedherein when used in combination with at least a first or second compoundof the invention.

Compounds of the invention can be prepared in a formulation orpharmaceutical composition appropriate for nasal administration. In someembodiments, the compounds of the invention can be formulated with amucosal penetration enhancer to facilitate delivery of the drug. Theformulation can also be prepared with pH optimized for solubility, drugstability, absorption through nasal mucosa, and other considerations.

Capsules, blisters, and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the pharmaceuticalcompositions provided herein; a suitable powder base, such as lactose orstarch; and a performance modifier, such as 1-leucine, mannitol, ormagnesium stearate. The lactose may be anhydrous or in the form of themonohydrate. Other suitable excipients include dextran, glucose,maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. Thepharmaceutical compositions provided herein for inhaled/intranasaladministration may further comprise a suitable flavor, such as mentholand levomenthol, or sweeteners, such as saccharin or saccharin sodium.

For administration by inhalation, the compounds for use according to themethods of the invention can be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the drugs and asuitable powder base such as lactose or starch.

The compounds may be administered to a subject as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc. For example, the site to be treated can becontacted with a composition once a day, twice a day, three times a day,four times a day. The composition can be contacted with the site atleast once a day for a period of 1-28 days, 1-21 days, 1-14 days, or 1-7days. In some embodiments, the composition is contacted with the site atleast once a day for at least 7, 14, 21, or 28 days.

Use of Cells

In some embodiments in which the treatment comprises cells include cellsthat can be cultured in vitro, derived from a natural source,genetically engineered, or produced by any other means.

In some embodiments the cells have been genetically engineered. Theengineering can involve programming the cell to express one or moregenes, repressing the expression of one or more genes, or both. Oneexample of genetically engineered cells useful in the present inventionis a genetically engineered cell that makes and secretes one or moredesired molecules. Cells may produce substances to aid in the followingnon-inclusive list of purposes: inhibit or stimulate inflammation;facilitate healing; resist immunorejection; provide hormone replacement;replace neurotransmitters; inhibit or destroy cancer cells; promote cellgrowth; inhibit or stimulate formation of blood vessels; augment tissue;and to supplement or replace neurons, skin, synovial fluid, tendons,cartilage (including, but not limited to articular cartilage),ligaments, bone, muscle, organs, dura, blood vessels, bone marrow, andextracellular matrix. Various growth factors, cytokines, or othermolecules may also be administered to regulate the cell and/or aid inthe function of interest of that cell.

The cells of the present invention may be administered to a subjectalone or in admixture with a composition useful in the repair of woundsand other defects. Such compositions include, but are not limited tobone morphogenetic proteins, hydroxyapatite/tricalcium phosphateparticles (HA/TCP), gelatin, poly-L-lysine, and collagen.

In some embodiments, cells of the invention can be used in conjunctionwith a product such as Dermagraft.® Dermagraft® is indicated for use inthe treatment of full-thickness diabetic foot ulcers greater than sixweeks duration, which extend through the dermis, but without tendon,muscle, joint capsule, or bone exposure. Dermagraft® is a cryopreservedhuman fibroblast-derived dermal substitute; it is composed offibroblasts, extracellular matrix, and a bioabsorbable scaffold.Dermagraft® is manufactured from human fibroblast cells derived fromnewborn foreskin tissue. During the manufacturing process, the humanfibroblasts are seeded onto a bioabsorbable polyglactin mesh scaffold.The fibroblasts proliferate to fill the interstices of this scaffold andsecrete human dermal collagen, matrix proteins, growth factors, andcytokines to create a three-dimensional human dermal substitutecontaining metabolically active living cells. Dermagraft® does notcontain macrophages, lymphocytes, blood vessels, or hair follicles.

In some embodiments, the invention provides a method of promoting theclosure of a wound within a subject using cells and compositions asdescribed herein. In accordance with the method, the inventive cellswhich have been selected or have been modified to secrete a hormone,growth factor, or other agent are transferred to the vicinity of a woundunder conditions sufficient for the cell to produce the hormone, growthfactor or other agent. The presence of the hormone, growth factor, orother agent in the vicinity of the wound promotes closure of the wound.In some embodiments, proliferation of the administered cells promoteshealing of the wound. In some embodiments, differentiation of theadministered cells promotes healing of the wound. The method promotesclosure of both external (e.g., surface) and internal wounds. The methodneed not achieve complete healing or closure of the wound; it issufficient for the method to promote any degree of wound closure. Inthis respect, the method can be employed alone or as an adjunct to othermethods for healing wounded tissue.

The present invention encompasses a method of treating a disorderamenable to cell therapy comprising administering to the affectedsubject a therapeutically effective amount of the cells of theinvention.

In some embodiments, the cells are obtained and cultured in order toderive and store the cells for therapeutic uses using cell therapyshould the subject require, for example, disease therapy, tissue repair,transplantation, treatment of a cellular debilitation, or treatment ofcellular dysfunctions in the future.

In some embodiments, cells derived from a subject are directlydifferentiated in vitro or in vivo to generate differentiating ordifferentiated cells without generating a cell line.

Such cell therapy methods encompass the use of the cells of thisinvention in combination with growth factors or chemokines such as thoseinducting proliferation, lineage-commitment, or genes or proteins ofinterest. Treatment methods may include providing stem or appropriateprecursor cells directly for transplantation where the tissue isregenerated in vivo or recreating the desired tissue in vitro and thenproviding the tissue to the affected subject.

The composites and/or cells of the present invention can be used as avehicle for the in situ delivery of biologically active agents. Thebiologically active agents incorporated into, or included as an additivewithin, the composite of the subject invention can include, withoutlimitation, medicaments, growth factors, vitamins, mineral supplements,substances used for the treatment, prevention, diagnosis, cure ormitigation of disease or illness, substances which affect the structureor function of the body, or drugs. The biologically active agents can beused, for example, to facilitate implantation of the composite or cellsuspension into a subject to promote subsequent integration and healingprocesses. The active agents include, but are not limited to, antifungalagents, antibacterial agents, anti-viral agents, anti-parasitic agents,growth factors, angiogenic factors, anesthetics, mucopolysaccharides,metals, cells, and other wound healing agents. Because the processingconditions can be relatively benign (physiological temperature and pH),live cells can be incorporated into the composite during its formation,or subsequently allowed to infiltrate the composite through tissueengineering techniques.

In some embodiments, a composition comprising the cells of the inventionis administered locally by injection. Compositions comprising the cellscan be further combined with known drugs, and in one embodiment, thedrugs are bound to the cells. These compositions can be prepared in theform of an implantable device that can be molded to a desired shape. Inone embodiment, a graft construct is prepared comprising a biocompatiblematrix and one or more cells of the present invention, wherein thematrix is formed in a shape to fill a gap or space created by theremoval of a tumor, injured, or diseased tissue.

The cells can be seeded onto the desired site within the tissue toestablish a population. Cells can be transferred to sites in vivo usingdevices such as catheters, trocars, cannulae, stents (which can beseeded with the cells), etc.

The cells can be employed alone or within biologically-compatiblecompositions to generate differentiated tissues and structures, both invivo and in vitro, or to stimulate a process of interest in a tissue.Additionally, the cells can be expanded and cultured to producehormones, growth factors, including pleiotropic growth factors,cytokines, and chemokines, and to provide conditioned culture media forsupporting the growth and expansion of other cell populations. Inanother aspect, the invention encompasses a lipo-derived latticesubstantially devoid of cells, which includes extracellular matrixmaterial form adipose tissue. The lattice can be used as a substrate tofacilitate the growth and differentiation of cells, whether in vivo orin vitro, into anlagen or mature tissue or structures, as well as toprovide an environment which maintains the viability of the cells.

The present invention thus provides methods and compositions fordelivering incredibly large numbers of ASCs, precursors, ordifferentiated cells derived from adipose tissue for the procedures andtreatments described herein. Additionally, for diseases that requirecell infusions or administration, adipose tissue harvest is minimallyinvasive, yields many cells, and can be done repeatedly

The present invention encompasses the preparation and use ofimmortalized cell lines, including, but not limited to, adiposetissue-derived cell lines capable of differentiating into at least onecell type. Various techniques for preparing immortalized cell lines areknown to those of ordinary skill in the art.

Compositions comprising cells of the invention can be employed in anysuitable manner to facilitate the growth and differentiation of thedesired tissue. For example, the composition can be constructed usingthree-dimensional or stereotactic modeling techniques. To direct thegrowth and differentiation of the desired structure, the composition canbe cultured ex vivo in a bioreactor or incubator, as appropriate. Insome embodiments, the structure is implanted within the host animaldirectly at the site in which it is desired to grow the tissue orstructure. In some embodiments, the composition can be engrafted onto ahost, where it will grow and mature until ready for use. Thereafter, themature structure (or anlage) is excised from the host and implanted intothe host, as appropriate.

One of ordinary skill in the art would appreciate that there are othercarriers useful for delivering the cells of the invention. Such carriersinclude, but are not limited to, calcium phosphate, hydroxyapatite, andsynthetic or natural polymers such as collagen or collagen fragments insoluble or aggregated forms. In some embodiments, such carriers serve todeliver the cells to a location or to several locations. In someembodiments, the carriers and cells can be delivered either throughsystemic administration or by implantation. Implantation can be into onesite or into several sites.

As indicated above, cells can be seeded onto and/or within theorganic/inorganic composites of the present invention. Likewise, tissuessuch as cartilage can be associated with the composites prior toimplantation within a patient. Examples of such cells include, but arenot limited to, bone cells (such as osteoclasts, osteoblasts, andosteocytes), blood cells, epithelial cells, neural cells (e.g., neurons,astrocytes, and oligodendrocytes), and dental cells (odontoblasts andameloblasts). Seeded cells can be autogenic, allogenic, or xenogeneic.Seeded cells can be encapsulated or non-encapsulated.

Additional Uses

In some embodiments, the pharmaceutical composition of the invention canbe used as a cleanser. In some embodiments, the composition can be usedas a wound cleanser or a skin cleanser. In some embodiments, the skin isinjured or diseased. One of ordinary skill in the art will understandthat the formulation used as a cleanser and its route and dosage ofadministration can be varied depending on the types of variablesdescribed herein.

Kits

The present invention should be construed to include kits for increasingoxygenation at a site of treatment. The invention includes a kit whichin some embodiments comprises a compound identified in the invention, astandard, other materials which are used to apply the invention and/ormanage the treatment site, and an instructional material which describesadministering the compound and any additional components to a cell or ananimal. This should be construed to include other embodiments of kitsthat are known to those skilled in the art, such as a kit comprising astandard and a (preferably sterile) solvent suitable for dissolving orsuspending the composition of the invention prior to administering thecompound to a cell or an animal. In some embodiments, the animal is amammal, such as a human.

The compositions can also comprise other ingredients. For example, thecomposition can comprise components as described in U.S. Publication No.US 20090202615, which is hereby incorporated by reference in itsentirety.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Thisinvention encompasses all combinations of the different aspects of theinvention noted herein. It is understood that any and all embodiments ofthe present invention may be taken in conjunction with any otherembodiment or embodiments to describe additional more preferredembodiments. It is also to be understood that each individual element ofthe preferred embodiments is intended to be taken individually as itsown independent preferred embodiment. Furthermore, any element of anembodiment is meant to be combined with any and all other elements fromany embodiment to describe an additional embodiment.

The examples provided throughout his application are non-inclusiveunless otherwise stated. They include but are not limited to the recitedexamples.

The previous description of the disclosed embodiments is provided toenable any person skilled in the art to make or use the presentinvention. Various modifications to these embodiments will be readilyapparent to those skilled in the art, and the generic principles definedherein may be applied to other embodiments without departing from thespirit or scope of the invention. Accordingly, the present invention isnot intended to be limited to the embodiments shown herein but is to beaccorded the widest scope consistent with the principles and novelfeatures disclosed herein.

Embodiments of the invention may also include formulations of poloxamerwith additional components, examples of which are disclosed in thefollowing tables. The invention is not limited to these examples. Forexample, in some embodiments, the composition or formulation comprisesthe formulation as disclosed in Formulations A, B, C, D, E, F, G, H, I,J, K, L, M, N, O, P, Q, R, S, T, U. These formulations can be preparedas liquids, gels or be impregnated into various fabrics, including, butnot limited to, dressings, wipes, and the like. The formulations can bealso be directly applied (topically) to the site as a liquid or a gel.The ingredients listed in the formulations described herein can becombined sequentially or simultaneously.

Formulation: Ingredients (range) A B C D E F G Water alone or withadditional — — — — — — — ingredients (balance) Aloe Barbadensis LeafExtract  17% 15.33%  13.67%   12% 10.33%  8.67%   7% (0-30%)Linoleamidopropyl Pg- 1.33% 1.33% 1.33% 1.33% 1.33% 1.33% 1.33% DimoniumChloride Phosphate (0-20%) Polysorbate 20 (0-10%) 0.44% 0.44% 0.44%0.44% 0.44% 0.44% 0.44% Sodium Coco-Pg Dimonium 0.44% 0.44% 0.44% 0.44%0.44% 0.44% 0.44% Chloride Phosphate (0-30%) Allantoin (0-20%) 0.36%0.36% 0.36% 0.36% 0.36% 0.36% 0.36% Disodium EDTA (0-5%) 0.18% 0.18%0.18% 0.18% 0.18% 0.18% 0.18% Sodium Benzoate (0-5%) 0.13% 0.13% 0.13%0.13% 0.13% 0.13% 0.13% Benzalkonium Chloride (0-5%) 0.10% 0.10% 0.10%0.10% 0.10% 0.10% 0.10% Fragrance (0-5%) 0.06% 0.06% 0.06% 0.06% 0.06%0.06% 0.06% Iodopropynyl Butylcarbamate 0.17% 0.17% 0.17% 0.17% 0.17%0.17% 0.17% (0-5%) Poloxamer 188 (0.01-75%) 75.0%  70%  65%  60%  55% 50%  45% Formulation: Ingredients H I J K L M N Water alone or withadditional — — — — — — — ingredients (balance) Aloe Barbadensis LeafExtract 5.33% 5.33%   2%   2% 3.67% 5.33% 7.00% (0-30%)Linoleamidopropyl Pg- 1.33% 1.33% 1.33% 1.33% — 12.33%   15% DimoniumChloride Phosphate (0-20%) Polysorbate 20 (0-10%) 0.44% 0.44% 0.44%0.44% 0.44% 0.44% 0.44% Sodium Coco-Pg Dimonium 0.44% 0.44% 0.44% 0.44%0.44% 0.44% 0.44% Chloride Phosphate (0-30%) Allantoin (0-20%) 0.36%0.36% 0.36%   2%   2%   2%   2% Disodium EDTA (0-5%) 0.18% 0.18% 0.18%0.18% 0.18% 0.18% 0.18% Sodium Benzoate (0-5%) 0.13% 0.13% 0.13% 0.13%0.13% 0.13% 0.13% Benzalkonium Chloride (0-5%)  0.1%  0.2%  0.3%  0.4% 0.5%   1%   2% Fragrance (0-5%) 0.06% 0.06% 0.06% — — — — IodopropynylButylcarbamate 0.17% 0.17% 0.17% 0.15% 0.15% 0.15% 0.15% (0-5%)Poloxamer 188 (0.01-75%)   5%   5%  0.1% 40.0%  35%  30%  25%Formulation: Ingredients O P Q R S T U Water alone or with additional —— — — — — — ingredients (balance) Aloe Barbadensis Leaf Extract 8.67%10.33%     12% 30% 15.33%   17% 20% (0-30%) Linoleamidopropyl Pg-   1%5% 7.5% 20%  15% 17.5% 10% Dimonium Chloride Phosphate (0-20%)Polysorbate 20 (0-10%)  0.1% 1%  4%  6%  8%   9% 10% Sodium Coco-PgDimonium   1% 5% 7.5% 10%  15% 17.5% 20% Chloride Phosphate (0-30%)Allantoin (0-20%)   2% 4%  8% 20%  15%  18% 12% Disodium EDTA (0-5%)0.15% 0.8%  1.5%  2%  3%  4.5%  5% Sodium Benzoate (0-5%)  0.1% 0.15%   2% 3.13%  3.38%    4%  5% Benzalkonium Chloride (0-5%) 0.15% 0.5% 0.75%  1.5%  1.5% 1.75%  2% Fragrance (0-5%) — — — — — — — IodopropynylButylcarbamate 0.15% 0.5%  0.75%  1.5%  1.5% 1.75%  2% (0-5%) Poloxamer188 (0.01-75%)  20% 15%   10%  5% 2.5%   1%  1%

EXAMPLE Example 1

A patient with a wound is cleansed with a wipe comprising a compositioncomprising a formulation selected from Formulations A-U. The wipe isimpregnated with the formulation. The treatment lasts two weeks.Bacterial population are evaluated by taking samples by plug, the woundarea reduction is visualized using the Visitrak™ system and thevariation in oxygen saturimetry is determined using a reflectancesaturimeter. The results demonstrate significant clinical improvement,particularly in terms of oxygen saturimetry improvement. Oxygensaturation increases by at least 5-10%. Bacterial count is also reduced.Cleansing and hydration of the perilesional area and border of the woundwith the wipe contributes to the healing process. This is determined byevaluating the level of oxygen in the perilesional tissue areas. Anincrease of the level of oxygen in the perilesional area is recorded.The efficacy in the reduction of the bacterial count is satisfactory. Incolonized wounds, significant clinical improvement are recorded,comparable in percentage terms to the improvements that are recorded onclean wounds. The activity of the formulation is evident from theclinical evaluation and from the oxygen levels that are recorded in theperilesional areas. Without wishing to be bound by theory, it ishypothesized that the surfactant component acts as an oxygen reservoirand/or delivery vehicle to increase oxygenation, reduce bacteria, andimprove healing.

1. A method for treating a wound, comprising: topically administrating acomposition comprising at least one surface active agent and about 10%to about 50% of a standard therapeutically effective amount of at leastone therapeutic agent selected from the group consisting ofantimicrobial agents, analgesics, and combinations thereof to the wound.2. The method of claim 1, wherein the composition is in the form of agel.
 3. The method of claim 1, wherein contacting comprises applying afabric material comprising the composition to the wound.
 4. The methodof claim 1, wherein the one or more surface active agents comprise asurface active copolymer.
 5. The method of claim 1, wherein topicallyadministrating comprises applying a fabric material comprising thecomposition to the wound.
 6. The method of claim 1, wherein the one ormore surface active agents comprise a surface active copolymer.
 7. Themethod of claim 1, further comprising contacting the site with a secondcomposition comprising a second surface active agent and at least oneadditional therapeutic agent.
 8. The method of claim 1, wherein the atleast one surface active agent is selected from the group consisting ofpoloxamers, meroxapols, poloxamines, and combinations thereof.
 9. Themethod of claim 1, wherein the at least one surface active agent isselected from the group consisting of poloxamer 127, poloxamer 188,poloxamer 237, poloxamer 335, poloxamer 407, and combinations thereof.10. The method of claim 1, wherein the at least one surface active agentcomprises about 0.01 w/w % to about 85 w/w %.
 11. The method of claim 1,wherein the antimicrobial agent comprises about 10% to about 50% of astandard therapeutically effective amount.
 12. The method of claim 1,wherein the at least one therapeutic agent is silver sulfadiazine, lessthan 1.0 w/w %.